Human sarcoma-associated antigens

ABSTRACT

The invention relates to sarcoma-associated antigens and the nucleic acid molecules that encode them. The invention further relates to the use of the nucleic acid molecules, polypeptides and fragments thereof associated with sarcoma in methods and compositions for the diagnosis and treatment of diseases, such as cancer. More specifically, the invention relates to the discovery of a novel cancer/testis (CT) antigen, NY-SAR-35.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/260,708, filed on Sep. 30, 2002, now pending, the disclosure of whichis incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to sarcoma-associated antigens and the nucleicacid molecules that encode them. The invention further relates to theuse of the nucleic acid molecules, polypeptides and fragments thereofassociated with sarcoma in methods and compositions for the diagnosisand treatment of diseases, such as cancer. More specifically, theinvention relates to the discovery of a novel cancer/testis (CT)antigen, NY-SAR-35.

BACKGROUND OF THE INVENTION

The identification of human tumor antigens recognized by the autologoushost is yielding new and promising target molecules for immunotherapy,diagnosis and monitoring of human cancer (van der Bruggen P, et al.1991. A gene encoding an antigen recognized by cytolytic T lymphocyteson a human melanoma. Science 254:1643-47; Gaugler, B., et al. Human geneMAGE-3 codes for an antigen recognized on a melanoma by autologouscytolytic T lymphocytes. J. Exp. Med. 1994; 179: 921-30; Kawakami, Y.,et al. Cloning of the gene for a shared human melanoma antigenrecognized by autologous T cells infiltrating into tumor. Proc. Natl.Acad. Sci. USA. 1994; 91: 3515-19 and Chen, Y.-T., et al. A testicularantigen aberrantly expressed in human cancers detected by autologousantibody screening. Proc. Natl. Acad. Sci. USA. 1997; 94: 1914-18).Studies of the cellular and humoral immune response to cancer haverevealed an extensive repertoire of tumor antigens recognized by theimmune system, collectively termed the cancer immunome (Jager D, et al.Identification of a tissue-specific putative transcription factor inbreast tissue by serological screening of a breast cancer library.Cancer Res 2001 Mar. 1; 61(5):2055-61).

The immunome is composed largely of antigens defined by T-cell epitopecloning (van der Bruggen P, et al. 1991. A gene encoding an antigenrecognized by cytolytic T lymphocytes on a human melanoma. Science254:1643-47; Gaugler, B., et al. Human gene MAGE-3 codes for an antigenrecognized on a melanoma by autologous cytolytic T lymphocytes. J. Exp.Med. 1994; 179: 921-30; Kawakami, et al. Cloning of the gene for ashared human melanoma antigen recognized by autologous T cellsinfiltrating into tumor. Proc. Natl. Acad. Sci. USA. 1994; 91: 3515-19;Boel, P., et al. BAGE: a new gene encoding an antigen recognized onhuman melanomas by cytolytic T lymphocytes. Immunity 1995; 2: 167-75.(PMID: 7895173); Van den Eynde, B., et al. A new family of genes codingfor an antigen recognized by autologous cytolytic T lymphocytes on ahuman melanoma. J. Exp. Med. 1995; 182: 689-98. (PMID: 7544395)), MHCpeptide elution (Skipper J C, et al. An HLA-A2-restricted tyrosinaseantigen on melanoma cells results from posttranslational modificationand suggests a novel pathway for processing of membrane proteins. J ExpMed 1996 Feb. 1; 183(2):527-34; Cox A L, et al. Identification of apeptide recognized by five melanoma-specific human cytotoxic T celllines. Science 1994 Apr. 29; 264(5159):716-9; Pascolo S, et al. AMAGE-A1 HLA-A A*0201 epitope identified by mass spectrometry. Cancer Res2001 May 15; 61(10):4072-7), and serological expression cloning (SEREX,Chen, Y.-T., et al. A testicular antigen aberrantly expressed in humancancers detected by autologous antibody screening. Proc. Natl. Acad.Sci. USA. 1997; 94: 1914-18; Jager D, et al. Identification of atissue-specific putative transcription factor in breast tissue byserological screening of a breast cancer library. Cancer Res 2001 Mar.1; 61(5):2055-61; Sahin, U., et al. Human neoplasms elicit multiplespecific immune responses in the autologous host. Proc. Natl. Acad. Sci.USA 1995; 92: 11810-13; Scanlan, M. J., et al. Characterization of humancolon cancer antigens recognized by autologous antibodies. Int. J.Cancer 1998; 76: 652-8; Scanlan, M. J., et al. Antigens recognized byautologous antibody in patients with renal-cell carcinoma. Int. J.Cancer 1999; 83: 456-64; Scanlan M J, et al. Humoral immunity to humanbreast cancer: antigen definition and quantitative analysis of mRNAexpression. Cancer Immunity 1:4 [epub]), and is catalogued in threedatabases: the peptide database of T-cell defined tumor antigens(authored by members of the Ludwig Institute for Cancer Research (LICR)that is available on the website of Cancer Immunity, Journal of theAcademy of Cancer Immunology,cancerimmunity.org/peptidedatabase/Tcellepitopes); the SYFPEITHIdatabase of MHC ligands and peptide motifs (available on the website ofBiomedical Informatics-Heidelberg, bmi-heidelberg.com/syfpeithi/) andthe cancer immunome database available on the website of the LICR(licr.org/CancerImmunomeDB, formerly licr.org/SEREX.html).

SEREX is a method of immunoscreening tumor-derived cDNA expressionlibraries with cancer patient sera in order to identify moleculesrecognized by high titered IgG antibodies (Sahin, U., et al. Humanneoplasms elicit multiple specific immune responses in the autologoushost. Proc. Natl. Acad. Sci. USA 1995; 92: 11810-13) Approximately 1000distinct antigens have been defined by SEREX analysis, including anumber of etiologically and therapeutically significant cancer antigens,such as mutational antigens (e.g. p53, LKB1, BUB1; Scanlan, M. J., etal. Characterization of human colon cancer antigens recognized byautologous antibodies. Int. J. Cancer 1998; 76: 652-8; Scanlan, M. J.,et al. Antigens recognized by autologous antibody in patients withrenal-cell carcinoma. Int. J. Cancer 1999; 83: 456-64; Scanlan M J, etal. Humoral immunity to human breast cancer: antigen definition andquantitative analysis of mRNA expression. Cancer Immunity 1:4 [epub]),differentiation antigens (e.g. tyrosinase, NY-BR-1, rab 38; Jager D, etal. Identification of a tissue-specific putative transcription factor inbreast tissue by serological screening of a breast cancer library.Cancer Res 2001 Mar. 1; 61(5):2055-61; Sahin, U., et al. Human neoplasmselicit multiple specific immune responses in the autologous host. Proc.Natl. Acad. Sci. USA 1995; 92: 11810-13; Jager D, et al. Serologicalcloning of a melanocyte rab guanosine 5′-triphosphate-binding proteinand a chromosome condensation protein from a melanoma complementary DNAlibrary. Cancer Res 2000 Jul. 1; 60(13):3584-91), overexpressed geneproducts (e.g. Her2neu, TPD52, eIF4-gamma; Scanlan M J, et al. Humoralimmunity to human breast cancer: antigen definition and quantitativeanalysis of mRNA expression. Cancer Immunity 1:4 [epub]; Chen, Y.-T., etal. Identification of human tumor antigens by serological expressioncloning. In: S. A. Rosenberg (ed.). Principles and Practice of BiologicTherapy of Cancer, pp. 557-570. Philadelphia: Lippincott Williams &Wilkins, 2000) and cancer/testis (CT) antigens (e.g. MAGE-1, NY-ESO-1,SSX-2; Chen, Y.-T., et al. A testicular antigen aberrantly expressed inhuman cancers detected by autologous antibody screening. Proc. Natl.Acad. Sci. USA. 1997; 94: 1914-18; Sahin, U., et al. Human neoplasmselicit multiple specific immune responses in the autologous host. Proc.Natl. Acad. Sci. USA 1995; 92: 11810-13).

CT antigens represent a group of shared, tumor-specific antigensexpressed exclusively in developing germ cells of the testis and fetalovary, as well as in placental trophoblast, and most notably, in aproportion of human cancers of diverse origins (Chen, Y.-T., et al.Identification of human tumor antigens by serological expressioncloning. In: S. A. Rosenberg (ed.). Principles and Practice of BiologicTherapy of Cancer, pp. 557-570. Philadelphia: Lippincott Williams &Wilkins, 2000). These antigens elicit spontaneous cellular (Van denEynde, B. J. and van der Bruggen, P. (1997) Curr. Opin. Immunol. 9,684-693) and humoral immune responses (Stockert, E., et al. (1998) J.Exp. Med. 187, 1349-1354) in some cancer patients. On the basis oftissue-restricted expression and immunogenicity, CT antigens areattractive targets for vaccine-based immunotherapies. In general, CTantigens are expressed in 20-40% of specimens from a given tumor type(Sahin U, et al. 1998. Expression of multiple cancer/testis antigens inbreast cancer and melanoma: basis for polyvalent CT vaccine strategies.Int J Cancer 78:387-89; Scanlan M J et al. 2000. Expression ofcancer-testis antigens in lung cancer: definition of bromodomaintestis-specific gene (BRDT) as a new CT gene, CT9. Cancer Lett.150:155-64; Van den Eynde B J and van der Bruggen P. 1997. T celldefined tumor antigens. Curr Opin Immunol 9:684-693). One exception tothis is synovial sarcoma, in which 80% of specimens express NY-ESO-1(Jungbluth A A, et al. 2001. Monophasic and biphasic synovial sarcomasabundantly express cancer/testis antigen NY-ESO-1 but not MAGE-A1 orCT7. Int J Cancer 94:252-6) and MAGE antigens (Antonescu C R, et al.MAGE antigen expression in monophasic and biphasic synovial sarcoma. HumPathol 2002 February; 33(2):225-9); the expression of which are oftenhomogeneous throughout the tumor. Thus, identification of additional CTantigens and other genes having a tumor-associated expression profile isneeded for the development of additional therapeutics and diagnostics topermit effective treatment and diagnosis of a broader group of cancerpatients.

SUMMARY OF THE INVENTION

The humoral immune response of sarcoma patients to CT antigens wasexamined using the SEREX method. Sera from patients which showed ahumoral immune response to CT antigens were subsequently used to screencDNA libraries derived from CT-rich synovial sarcoma cell lines as wellas normal testis. Although there was little overlap in the identity ofclones isolated with different sarcoma sera, more than 30% of theisolated clones were previously identified during SEREX analysis ofother tumor types. Approximately 60% of these antigens also reacted withsera from normal individuals. This is in conformity with other findings(Scanlan, M. J., et al. Antigens recognized by autologous antibody inpatients with renal-cell carcinoma. Int. J. Cancer 1999; 83: 456-64 andScanlan M J, et al. Humoral immunity to human breast cancer: antigendefinition and quantitative analysis of mRNA expression. Cancer Immunity20001; 1:4 [epub]). Thus, only a fraction of the serologically-definedimmunome is associated with a cancer-related immune response. Thestudies described herein have led to the identification of antigens,which include antigens not before associated with cancer along withseveral novel gene products associated with a sarcoma-related immuneresponse. One such novel CT antigen is NY-SAR-35, which appears to be acell surface/secreted molecule.

According to one aspect of the invention, isolated nucleic acidmolecules are provided. The isolated nucleic acid molecules are selectedfrom the group consisting of (a) nucleic acid molecules which hybridizeunder high stringency conditions to a nucleic acid molecule comprising anucleotide sequence selected from the group consisting of nucleotidesequences set forth as SEQ ID NOs: 1-14 and 97-107 and which code for asarcoma-associated antigen, (b) nucleic acid molecules that differ fromthe nucleic acid molecules of (a) in codon sequence due to thedegeneracy of the genetic code, and (c) complements of (a) or (b).

In some embodiments, the isolated nucleic acid molecule includes anucleotide sequence selected from the group consisting of nucleotidesequences set forth as SEQ ID NOs: 1-14 and 97-107. In some embodimentsthe isolated nucleic acid molecule comprises a nucleotide sequenceselected from the group consisting of nucleotide sequences set forth asSEQ ID NOs: 10, 11, 99, 102 and 104. In other embodiments the nucleicacid molecule comprises a nucleotide sequence set forth as SEQ ID NO:10. In yet other embodiments the nucleic acid molecule comprises anucleotide sequence set forth as SEQ ID NO: 11. In still otherembodiments the nucleic acid molecule comprises a nucleotide sequenceset forth as SEQ ID NO: 102. In still further embodiments the nucleicacid molecule comprises a nucleotide sequence set forth as SEQ ID NO:104.

In some embodiments the nucleic acid molecule comprises a nucleotidesequence set forth as SEQ ID NOs: 121, 123, 125, 127, 129 or 131. Insome embodiments the nucleic acid molecule comprises a nucleotidesequence set forth as SEQ ID NO: 121. In other embodiments the nucleicacid molecule comprises a nucleotide sequence set forth as SEQ ID NO:123. In still other embodiments the nucleic acid molecule comprises anucleotide sequence set forth as SEQ ID NO: 125. In yet otherembodiments the nucleic acid molecule comprises a nucleotide sequenceset forth as SEQ ID NO: 131.

According to another aspect of the invention, additional isolatednucleic acid molecules are provided. The isolated nucleic acid moleculesare selected from the group consisting of: (a) unique fragments of anucleotide sequence selected from the group consisting of nucleotidesequences set forth as SEQ ID NOs: 10, 11, 99, 102 and 104, whichencodes an immunogenic peptide and (b) complements of (a). In someembodiments the isolated nucleic acid molecules are selected from thegroup consisting of: (a) unique fragments of a nucleotide sequence setforth as SEQ ID NO: 10, which encodes an immunogenic peptide and (b)complements of (a). In other embodiments the isolated nucleic acidmolecules are selected from the group consisting of: (a) uniquefragments of a nucleotide sequence set forth as SEQ ID NO: 11, whichencodes an immunogenic peptide and (b) complements of (a). In yet otherembodiments the isolated nucleic acid molecules are selected from thegroup consisting of: (a) unique fragments of a nucleotide sequence setforth as SEQ ID NO: 102, which encodes an immunogenic peptide and (b)complements of (a). In still other embodiments the isolated nucleic acidmolecules are selected from the group consisting of: (a) uniquefragments of a nucleotide sequence set forth as SEQ ID NO: 104, whichencodes an immunogenic peptide and (b) complements of (a). In someembodiments the nucleic acid molecule comprises a nucleotide sequenceset forth as SEQ ID NO: 121. In other embodiments the nucleic acidmolecule comprises a nucleotide sequence set forth as SEQ ID NO: 123. Instill other embodiments the nucleic acid molecule comprises a nucleotidesequence set forth as SEQ ID NO: 125. In yet other embodiments thenucleic acid molecule comprises a nucleotide sequence set forth as SEQID NO: 131.

In certain embodiments, the isolated nucleic acid molecule includes anucleotide sequence that is at least about 90% identical to a nucleotidesequence selected from the group consisting of SEQ ID NOs: 1-14 and97-107; preferably the nucleotide sequence is at least about 95%identical, more preferably the nucleotide sequence is at least about 97%identical, still more preferably the nucleotide sequence is at leastabout 98% identical, and yet more preferably the nucleotide sequence isat least about 99% identical.

According to further aspects of the invention, expression vectors thatinclude any of the foregoing isolated nucleic acid molecules operablylinked to a promoter are provided, as are host cells transformed ortransfected with these expression vectors. In certain embodiments, thehost cell expresses a MHC molecule, and in some of these embodiments theMHC molecule is expressed recombinantly.

According to another aspect of the invention, isolated polypeptides areprovided that are encoded by the isolated nucleic acid moleculesdescribed herein. In certain embodiments, the isolated polypeptideincludes an amino acid sequence as set forth in SEQ ID NOs: 46-60,109-120 or a fragment thereof that is at least eight amino acids inlength. In certain embodiments, the isolated polypeptides are antigenicpolypeptides that are capable of eliciting antibodies to asarcoma-associated antigen. In some embodiments the isolated polypeptideincludes an amino acid sequence as set forth in SEQ ID NO: 55 or afragment thereof that is at least eight amino acids in length. In otherembodiments the isolated polypeptide includes an amino acid sequence asset forth in SEQ ID NO: 56 or a fragment thereof that is at least eightamino acids in length. In yet other embodiments the isolated polypeptideincludes an amino acid sequence as set forth in SEQ ID NO: 111 or afragment thereof that is at least eight amino acids in length. In stillother embodiments the isolated polypeptide includes an amino acidsequence as set forth in SEQ ID NO: 114 or a fragment thereof that is atleast eight amino acids in length. In yet other embodiments the isolatedpolypeptide includes an amino acid sequence as set forth in SEQ ID NO:116 or a fragment thereof that is at least eight amino acids in length.In still other embodiments the isolated polypeptide includes an aminoacid sequence as set forth in SEQ ID NO: 122. In yet other embodimentsthe isolated polypeptide includes an amino acid sequence as set forth inSEQ ID NO: 124. In still further embodiments the isolated polypeptideincludes an amino acid sequence as set forth in SEQ ID NO: 126. In yetother embodiments the polypeptide includes the amino acid sequence setforth as SEQ ID NOs: 128, 130 or 132.

Another aspect of the invention provides binding polypeptides thatselectively bind to the foregoing isolated polypeptides. In someembodiments these binding polypeptides are isolated also. In otherembodiments, the binding polypeptides are antibodies or antigen-bindingfragments thereof.

According to another aspect of the invention, methods of diagnosingcancer in a subject are provided. The methods include obtaining abiological sample from the subject, and determining the presence of anantibody in the biological sample that binds specifically to one or moresarcoma-associated antigens encoded by a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and108. The presence of such antibodies indicates that the subject hascancer. In some embodiments the one or more sarcoma-associated antigensis/are encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 5-7, 10-13, 15-45, 102, 104 and 108. In stillother embodiments the one or more sarcoma-associated antigens is/areencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 10, 11, 15, 102, 104 and 108. In some embodiments thesarcoma-associated antigens is encoded by a nucleotide sequence setforth as SEQ ID NO: 10.

In some embodiments, the step of determining the presence of an antibodyincludes contacting the biological sample with one or moresarcoma-associated antigens that are specifically bound by the antibodyand are encoded by a nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of (1) nucleotide sequencesset forth as SEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and 108 and (2)nucleotide sequences that are at least 90% identical to the nucleotidesequences of (1), and then determining the binding of the antibody tothe sarcoma-associated antigen. In other embodiments, the step ofdetermining the presence of an antibody includes contacting thebiological sample with one or more sarcoma-associated antigens that arespecifically bound by the antibody and are encoded by a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of (1) nucleotide sequences set forth as SEQ ID NOs: 5-7,10-13, 15-45, 102, 104 and 108 and (2) nucleotide sequences that are atleast 90% identical to the nucleotide sequences of (1), and thendetermining the binding of the antibody to the sarcoma-associatedantigen.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence selected from the group consisting of nucleotide sequences setforth as SEQ ID NOs: 10, 11, 15, 102, 104 and 108, and in otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth as SEQ ID NO: 10. In other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth as SEQ ID NO: 121.In still other embodiments the nucleic acid molecule includes thenucleotide sequence set forth in SEQ ID NO: 123. In other embodimentsthe nucleic acid molecule includes the nucleotide sequence set forth inSEQ ID NO: 125. In yet other embodiments the nucleic acid moleculeincludes the nucleotide sequence set forth in SEQ ID NOs: 127, 129 or131.

In other embodiments, the sarcoma-associated antigen is a polypeptidethat includes the amino acid sequence of any of SEQ ID NOs: 48, 50-53,55-90, 111, 114, 116 and 120 or a fragment thereof that is at leasteight amino acids in length. In still other embodiments, thesarcoma-associated antigen is a polypeptide that includes the amino acidsequence of any of SEQ ID NOs: 50-52, 55-58, 60-90, 114, 116, 120 or afragment thereof that is at least eight amino acids in length. In stillother embodiments, the sarcoma-associated antigen is a polypeptide thatincludes the amino acid sequence of any of SEQ ID NOs: 55, 56, 60, 114,116 and 120 or a fragment thereof that is at least eight amino acids inlength.

In some embodiments, the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 55 or a fragment thereof that isat least eight amino acids in length. In certain embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 122. In still other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NO: 124. Inyet other embodiments the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 126. In still other embodimentsthe sarcoma-associated antigen includes the amino acid sequence setforth as SEQ ID NO: 128, 130 or 132.

In certain embodiments, the biological sample is serum. In otherembodiments, the one or more sarcoma-associated antigens are producedrecombinantly, and/or the one or more sarcoma-associated antigens arebound to a substrate. In some embodiments, the step of determining thebinding of the antibody with the one or more sarcoma-associated antigensis performed with an ELISA-based method. In still other embodiments aserum antibody detection assay (SADA) is used.

According to still another aspect of the invention, methods fordiagnosing cancer in a subject are provided. The methods includeobtaining a biological sample from a subject, and determining theexpression of a sarcoma-associated antigen or a nucleic acid moleculethat encodes it. The nucleic acid molecule includes a nucleotidesequence selected from the group consisting of SEQ ID NOs: 3, 5-8,10-45, 99, 102, 104 and 108 in the biological sample. The nucleic acidmolecule is some embodiments includes a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 5-7, 10-13, 15-45, 102, 104 and108 in the biological sample. The expression of the sarcoma-associatedantigen or the nucleic acid molecule that encodes it in the sample isdiagnostic for cancer in the subject.

In certain embodiments, the sarcoma-associated nucleic acid moleculecomprises the nucleotide sequence selected from the group consisting ofSEQ ID NOs: 10, 11, 15, 102, 104 and 108. In some embodiments thesarcoma-associated nucleic acid molecule includes the nucleotidesequence set forth as SEQ ID NO: 10. In other embodiments the nucleicacid molecule includes the nucleotide sequence set forth as SEQ ID NO:121. In still other embodiments the nucleic acid molecule includes thenucleotide sequence set forth in SEQ ID NO: 123. In yet otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth in SEQ ID NO: 125. In yet other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth in SEQ ID NOs: 127,129 or 131.

In other embodiments, the sarcoma-associated antigen comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 48,50-53, 55-90, 111, 114, 116 and 120 or a fragment thereof that is atleast eight amino acids in length. In yet other embodiments, thesarcoma-associated antigen comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 50-52, 55-58, 60-90, 114, 116and 120 or a fragment thereof that is at least eight amino acids inlength. The sarcoma-associated antigen in some embodiments includes anamino acid sequence selected from the group consisting of SEQ ID NOs:55, 56, 60, 114, 116 and 120. In some embodiments the sarcoma-associatedantigen includes an amino acid sequence set forth as SEQ ID NO: 55 or afragment thereof that is at least eight amino acids in length. Incertain embodiments the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 122. In still other embodimentsthe sarcoma-associated antigen includes the amino acid sequence setforth as SEQ ID NO: 124. In yet other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NO: 126. Instill further embodiments the sarcoma-associated antigen includes theamino acid sequence set forth as SEQ ID NOs: 128, 130 or 132.

According to yet another aspect of the invention, methods fordetermining onset, progression, or regression of cancer in a subject areprovided. The methods include obtaining from a subject a firstbiological sample, determining the expression of a sarcoma-associatedantigen or the nucleic acid molecule that encodes it in the firstsample, obtaining from the subject a second biological sample,determining the expression of the sarcoma-associated antigen or thenucleic acid molecule that encodes it in the second sample, andcomparing the expression in the first sample to the expression in thesecond sample as a determination of the onset, progression, orregression of the cancer. The nucleic acid molecule comprises anucleotide sequence selected from the group consisting of (1) nucleotidesequences set forth as SEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and 108and (2) nucleotide sequences that are at least 90% identical to thenucleotide sequences of (1). In some embodiments the nucleic acidmolecule comprises a nucleotide sequence selected from the groupconsisting of (1) nucleotide sequences set forth as SEQ ID NOs: 5-7,10-13, 15-45, 102, 104 and 108 and (2) nucleotide sequences that are atleast 90% identical to the nucleotide sequences of (1).

In some embodiments, the nucleic acid molecule that encodes thesarcoma-associated antigen includes a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 10, 11, 15, 102, 104 and 108. Inother embodiments the nucleic acid molecule includes the nucleotidesequence of SEQ ID NO: 10. In yet other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth as SEQ ID NO: 121.In still other embodiments the nucleic acid molecule includes thenucleotide sequence set forth in SEQ ID NO: 123. In yet otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth in SEQ ID NO: 125. In still other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth in SEQ ID NOs: 127,129 or 131. In other embodiments, the sarcoma-associated antigenincludes a polypeptide sequence selected from the group consisting ofpolypeptide sequences set forth as SEQ ID NOs: 48, 50-53, 55-90, 111,114, 116 and 120 or a fragment thereof that is at least eight aminoacids in length. In still other embodiments, the sarcoma-associatedantigen includes a polypeptide sequence selected from the groupconsisting of polypeptide sequences set forth as SEQ ID NOs: 50-52,55-58, 60-90, 114, 116 and 120 or a fragment thereof that is at leasteight amino acids in length. In yet other embodiments, thesarcoma-associated antigen includes a polypeptide sequence selected fromthe group consisting of polypeptide sequences set forth as SEQ ID NOs:55, 56, 60, 114, 116 and 120 or a fragment thereof that is at leasteight amino acids in length. In some embodiments the sarcoma-associatedantigen includes the amino acid sequence of SEQ ID NO: 55 or a fragmentthereof that is at least eight amino acids in length. In certainembodiments the sarcoma-associated antigen includes the amino acidsequence set forth as SEQ ID NO: 122. In still other embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 124. In yet other embodiments the sarcoma-associated antigenincludes the amino acid sequence set forth as SEQ ID NO: 126. In stillother embodiments the sarcoma-associated antigen includes the amino acidsequence set forth as SEQ ID NOs: 128, 130 or 132.

In some embodiments of the foregoing methods, the step of determiningthe expression of the sarcoma-associated antigen or the nucleic acidmolecule that encodes it includes contacting the biological sample withan agent that selectively binds to the sarcoma-associated antigen or thenucleic acid molecule that encodes it. For methods in which the agentthat selectively binds is a nucleic acid molecule, it is preferred thatthe expression of the sarcoma-associated nucleic acid molecule isdetermined by nucleic acid hybridization or nucleic acid amplification;some embodiments of the methods utilize real-time RT-PCR or RT-PCR asmethods of nucleic acid amplification, or use a nucleic acid microarrayas a method for nucleic acid hybridization. For methods in which theagent that selectively binds is a polypeptide, the polypeptidepreferably is an antibody or antigen-binding fragment thereof. Morepreferably, the antibody is a monoclonal antibody, particularly achimeric, human, or humanized antibody, a single chain antibody, or theantigen-binding fragment is a F(ab′)₂, Fab, Fd, or Fv fragment. Incertain embodiments, the antibody or antigen-binding fragment is labeledwith a detectable label, preferably a fluorescent or radioactive label.

In certain embodiments of the foregoing methods, the sample is selectedfrom the group consisting of tissue, cells, and blood. In someembodiments, the cancer is a sarcoma.

In another aspect of the invention, kits for detecting antibodiesreactive to a sarcoma-associated antigen in a biological sample areprovided. The kits include one or more sarcoma-associated antigensencoded by a nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of nucleotide sequences set forth asSEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and 108, and instructions forthe use of the sarcoma-associated antigens in the detection ofantibodies in the biological sample. In some embodiments the one or moresarcoma-associated antigens is/are encoded by a nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting ofnucleotide sequences set forth as SEQ ID NOs: 5-7, 10-13, 15-45, 102,104 and 108. In some embodiments, the sarcoma-associated nucleic acidmolecule comprises the nucleotide sequence set forth as SEQ ID NO: 10,11, 15, 102, 104 or 108. In other embodiments, the sarcoma-associatednucleic acid molecule comprises the nucleotide sequence set forth as SEQID NO: 10. In other embodiments the nucleic acid molecule includes thenucleotide sequence set forth as SEQ ID NO: 121. In still otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth in SEQ ID NO: 123. In yet other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth in SEQ ID NO: 125.In still further embodiments the nucleic acid molecule includes thenucleotide sequence set forth in SEQ ID NOs: 127, 129 or 131. In otherembodiments, the sarcoma-associated antigens are bound to a substrate.In further embodiments, the kit also includes a labeling reagent andlabeling reagent substrate, and/or a blocking reagent. Additional kitembodiments include secondary antibodies for detection of the antibodybound to the antigen.

In a further aspect of the invention, other kits for the diagnosis ofcancer in a subject are provided. The kits include one or more bindingagents that specifically bind to a sarcoma-associated antigen or thenucleic acid molecule that encodes it. In this aspect, the nucleic acidmolecule includes a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and 108. In someembodiments the nucleic acid molecule includes a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 5-7, 10-13, 15-45,102, 104 and 108. The kit also includes instructions for the use of thebinding agents in the diagnosis of cancer. The one or more bindingagents are nucleic acid molecules or polypeptides. If the latter, thepolypeptides preferably are antibodies or antigen-binding fragmentsthereof. In other embodiments, the one or more agents are bound to asubstrate. Further embodiments of the kits include one or more agentsthat bind specifically to a cancer-associated antigen other than thoseencoded by a nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 10, 11, 15, 102, 104and 108. In some embodiments, the kit is configured for diagnosis ofsarcomas.

According to another aspect of the invention, methods for treating asubject with a disorder characterized by the aberrant expression of asarcoma-associated antigen or the nucleic acid molecule that encodes itare provided. The methods include administering to a subject aneffective amount of an antibody or antigen-binding fragment thereof thatspecifically binds to the sarcoma-associated antigen. In this aspect,the antigen includes an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 48, 50-53, 55-90, 111, 114, 116 and 120 or afragment thereof that is eight or more amino acids in length. In someembodiments the antigen includes an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 50-52, 55-58, 60-90, 114, 116 and120 or a fragment thereof that is eight or more amino acids in length.In other embodiments the antigen includes an amino acid sequenceselected from the group consisting of SEQ ID NOs: 55, 56, 60, 114, 116and 120 or a fragment thereof that is eight or more amino acids inlength. In some embodiments, the antibody or antigen-binding fragmentthereof specifically binds to the extracellular domain of asarcoma-associated antigen that includes the amino acid sequence of SEQID NO: 55 or a fragment thereof that is eight or more amino acids inlength. In certain embodiments the sarcoma-associated antigen includesthe amino acid sequence set forth as SEQ ID NO: 122. In still otherembodiments the sarcoma-associated antigen includes the amino acidsequence set forth as SEQ ID NO: 124. In yet other embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 126. In still other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NO: 128,130 or 132. In yet other embodiments the sarcoma-associated antigenincludes the amino acid sequence set forth as SEQ ID NO: 134 or afragment thereof that is eight or more amino acids in length.

In certain embodiments, the disorder is cancer, preferably sarcoma. Inother embodiments, the antibody used in the methods is a monoclonalantibody, preferably a chimeric, human, or humanized antibody; a singlechain antibody; or the antigen-binding fragment is a F(ab′)₂, Fab, Fd,or Fv fragment.

In other embodiments, the antibody or antigen-binding fragment thereofis bound to a cytotoxic agent. Preferred cytotoxic agents include:calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan,chlorambucil, ARA-C, vindesine, mitomycin C, cisplatinum, etopside,bleomycin and 5-fluorouracil. Other cytotoxic agents includeradioisotopes, including those that emit α, β, and/or γ radiation.Preferred radioisotopes include: ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh,¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I, ¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu,²¹²Pb, ²²⁴Ra and ²²³Ra.

According to another aspect of the invention, methods for treating asubject with a disorder characterized by the aberrant expression of asarcoma-associated antigen or a nucleic acid molecule that encodes itare provided. The methods include administering an amount of an agentthat selectively binds to the sarcoma-associated antigen or the nucleicacid molecule that encodes it effective to treat the disorder. Thenucleic acid molecule comprises a nucleotide sequence selected from thegroup consisting of (a) an isolated nucleic acid molecule comprising anucleotide sequence that is at least 90% identical to the nucleotidesequence selected from the group consisting of SEQ ID NOs: 3, 5-8, 1045,99, 102, 104 and 108, and (b) nucleic acid molecules that differ fromthe nucleic acid molecules of (a) in codon sequence due to thedegeneracy of the genetic code. In some embodiments the nucleic acidmolecule comprises a nucleotide sequence selected from the groupconsisting of (a) an isolated nucleic acid molecule comprising anucleotide sequence that is at least 90% identical to the nucleotidesequence selected from the group consisting of SEQ ID NOs: 5-7, 10-13,15-45, 102, 104 and 108, and (b) nucleic acid molecules that differ fromthe nucleic acid molecules of (a) in codon sequence due to thedegeneracy of the genetic code. In certain embodiments the disorder iscancer, preferably sarcoma. In yet other embodiments thesarcoma-associated nucleic acid molecule comprises the nucleotidesequence selected from the group consisting of nucleotide sequences setforth as SEQ ID NOs: 10, 11, 15, 102, 104 and 108. In some embodimentsthe sarcoma-associated nucleic acid molecule comprises the nucleotidesequence set forth as SEQ ID NO: 10.

In other embodiments the sarcoma-associated nucleic acid molecule codesfor a sarcoma-associated antigen which comprises the polypeptidesequence selected from the group consisting of polypeptide sequences setforth as SEQ ID NOs: 48, 50-53, 55-90, 111, 114, 116 and 120 or afragment thereof that is at least eight amino acids in length. In stillother embodiments the sarcoma-associated nucleic acid molecule codes fora sarcoma-associated antigen which comprises the polypeptide sequenceselected from the group consisting of polypeptide sequences set forth asSEQ ID NOs: 50-52, 55-58, 60-90, 114, 116 and 120 or a fragment thereofthat is at least eight amino acids in length. In some embodiments thesarcoma-associated nucleic acid molecule codes for a sarcoma-associatedantigen which comprises the polypeptide sequence set forth as SEQ ID NO:55, 56, 60, 114, 116 or 120 or a fragment thereof that is at least eightamino acids in length. In another embodiment the sarcoma-associatednucleic acid molecule codes for a sarcoma-associated antigen whichcomprises the polypeptide sequence set forth as SEQ ID NO: 55 or afragment thereof that is at least eight amino acids in length. Incertain embodiments the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 122. In still other embodimentsthe sarcoma-associated antigen includes the amino acid sequence setforth as SEQ ID NO: 124. In yet other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NO: 126. Instill other embodiments the sarcoma-associated antigen includes theamino acid sequence set forth as SEQ ID NOs: 128, 130 or 132.

In certain embodiments, the binding agent is an antisense or RNAimolecule. In other embodiments, the binding agent is a polypeptide,preferably an antibody or antigen-binding fragment thereof. Preferredantibodies include monoclonal antibodies, including chimeric, human, orhumanized antibodies, and single chain antibodies; preferredantigen-binding fragments include F(ab′)₂, Fab, Pd, or Fv fragments. Inother embodiments, the antibody or antigen-binding fragment is bound toa cytotoxic agent.

According to yet another aspect of the invention, methods for treating asubject with a disorder characterized by the aberrant expression of asarcoma-associated antigen or the nucleic acid molecule that encodes itare provided. The methods include administering to the subject an amountof an agent effective to stimulate an immune response to asarcoma-associated antigen encoded by a nucleic acid molecule comprisinga nucleotide sequence that is at least 90% identical to the nucleotidesequence selected from the group consisting of SEQ ID NOs: 3, 5-8,10-45, 99, 102, 104 and 108. In some embodiments the sarcoma-associatedantigen is encoded by a nucleic acid molecule comprising a nucleotidesequence that is at least 90% identical to the nucleotide sequenceselected from the group consisting of SEQ ID NOs: 5-7, 10-13, 15-45,102, 104 and 108. In some embodiments, the disorder is cancer,particularly sarcoma. In other embodiments the sarcoma-associatednucleic acid molecule comprises the nucleotide sequence selected fromthe group consisting of nucleotide sequences set forth as SEQ ID NOs:10, 11, 15, 102, 104 and 108. In some embodiments the sarcoma-associatednucleic acid molecule comprises the nucleotide sequence set forth as SEQID NO: 10. In other embodiments the sarcoma-associated antigen isencoded by a nucleic acid molecule comprising a nucleotide sequence setforth as SEQ ID NO: 133.

In yet other embodiments the sarcoma-associated antigen includes anamino acid sequence selected from the group consisting of SEQ ID NOs:48, 50-53, 55-90, 111, 114, 116 and 120, or a fragment thereof that isat least eight amino acids in length. In still other embodiments thesarcoma-associated antigen includes an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 50-52, 55-58, 60-90, 114, 116 and120, or a fragment thereof that is at least eight amino acids in length.In some embodiments the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 55, or a fragment thereof that isat least eight amino acids in length. In certain embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 122. In still other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NO: 124. Inyet other embodiments the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 126. In still other embodimentsthe sarcoma-associated antigen includes the amino acid sequence setforth as SEQ ID NOs: 128, 130 or 132. In still further embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 134, or a fragment thereof that is at least eight amino acidsin length.

In some embodiments, the agent that stimulates an immune response is anucleic acid that encodes a sarcoma-associated antigen operably linkedto a promoter for expressing the sarcoma-associated antigen; apolypeptide comprising the sarcoma-associated antigen; or a host cellthat expresses the sarcoma-associated antigen, particularly a host cellthat also expresses a MHC molecule. In some embodiments, the agent whichstimulates an immune response is a peptide fragment of thesarcoma-associated antigen, or is a complex of a peptide fragment of thesarcoma-associated antigen and a MHC molecule. In other embodiments, theagent also includes an adjuvant or cytokine.

In another aspect of the invention, kits for diagnosing a disorderassociated with the aberrant expression of a sarcoma-associated antigenor a nucleic acid molecule that encodes it are provided. The kitsinclude one or more nucleic acid molecules that hybridize to the nucleicacid molecule that encodes the sarcoma-associated antigen comprising anucleotide sequence selected from the group consisting of nucleotidesequences set forth as SEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and 108under high stringency conditions, and instructions for the use of thenucleic acid molecules in the diagnosis of a disorder associated withaberrant expression of the sarcoma-associated antigen or the nucleicacid molecule that encodes it. In some embodiments the one or morenucleic acid molecules that hybridize to the nucleic acid molecule thatencodes the sarcoma-associated antigen comprises a nucleotide sequenceselected from the group consisting of nucleotide sequences set forth asSEQ ID NOs: 5-7, 10-13, 15-45, 102, 104 and 108. In some embodiments,the one or more nucleic acid molecules are detectably labeled. In someembodiments the nucleic acid molecule that encodes thesarcoma-associated antigen comprises the nucleotide sequence set forthas SEQ ID NO: 10, 11, 15, 102, 104 or 108. In other embodiments thenucleic acid molecule that encodes the sarcoma-associated antigencomprises the nucleotide sequence set forth as SEQ ID NO: 10. In otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth as SEQ ID NO: 121. In still other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth in SEQ ID NO: 123.In yet other embodiments the nucleic acid molecule includes thenucleotide sequence set forth in SEQ ID NO: 125. In still otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth in SEQ D NOs: 127, 129 or 131.

In certain embodiments, the one or more nucleic acid molecules consistof a first primer and a second primer, wherein the first primer and thesecond primer are constructed and arranged to selectively amplify atleast a portion of a nucleic acid molecule that encodes thesarcoma-associated antigen and comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 10, 11, 15, 102, 104 and 108.In other embodiments, the nucleic acids in the kit are bound to asubstrate.

In still another aspect of the invention, methods for identifying acancer-associated antigen are provided. The methods include obtaining abiological sample from one or more subjects, determining the reactivityof the biological sample to one or more known cancer-associatedantigens, using the reactive biological sample to screen an expressionlibrary to determine the presence of cancer-associated antigens reactivewith the biological sample, and isolating a clone that encodes thecancer-associated antigen from the expression library. In certainembodiments the biological sample is serum. In some embodiments theexpression library is derived from a tumor, preferably from a tumor cellline. In still other embodiments, the methods also include determiningthe identity of the cancer-associated antigen encoded by the isolatedclone, preferably by DNA sequencing.

The invention in a further aspect provides a composition including anagent that stimulates an immune response to a sarcoma-associatedantigen. In some embodiments sarcoma-associated antigens are thoseencoded by a nucleic acid molecule selected from the group consisting ofan isolated nucleic acid molecule comprising a nucleotide sequence thatis at least 90% identical to the nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 3, 5-8, 10-45, 99, 102, 104 and 108, andnucleic acid molecules that differ from the nucleic acid molecules of(a) in codon sequence due to the degeneracy of the genetic code. In someembodiments the sarcoma-associated antigens are those encoded by anucleic acid molecule selected from the group consisting of an isolatednucleic acid molecule comprising a nucleotide sequence that is at least90% identical to the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 5-7, 10-13, 15-45, 102, 104 and 108, andnucleic acid molecules that differ from the nucleic acid molecules of(a) in codon sequence due to the degeneracy of the genetic code. Inparticular embodiments, the nucleic acid molecule includes a nucleotidesequence selected from the group consisting of SEQ ID NOs: 10, 11, 15,102, 104 and 108. In some embodiments the nucleic acid molecule includesthe nucleotide sequence set forth as SEQ ID NO: 10. In other embodimentsthe nucleic acid molecule includes the nucleotide sequence set forth asSEQ ID NO: 133.

In some embodiments, sarcoma-associated antigen comprises a polypeptidesequence selected from the group consisting of SEQ ID NOs: 48, 50-53,55-90, 111, 114, 116 and 120 or a fragment thereof that is at leasteight amino acids in length. In other embodiments, sarcoma-associatedantigen comprises a polypeptide sequence selected from the groupconsisting of SEQ ID NOs: 50-52, 55-58, 60-90, 114, 116 and 120 or afragment thereof that is at least eight amino acids in length. In someembodiments the sarcoma-associated antigen includes the amino acidsequence of SEQ ID NO: 55, 56, 60, 114, 116 or 120 or a fragment thereofthat is at least eight amino acids in length. In other embodiments thesarcoma-associated antigen includes the amino acid sequence of SEQ IDNO: 55 or a fragment thereof that is at least eight amino acids inlength. In certain embodiments the sarcoma-associated antigen includesthe amino acid sequence set forth as SEQ ID NO: 122. In still otherembodiments the sarcoma-associated antigen includes the amino acidsequence set forth as SEQ ID NO: 124. In yet other embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 126. In still other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NOs: 128,130 or 132. In yet other embodiments the sarcoma-associated antigenincludes the amino acid sequence set forth as SEQ ID NO: 134.

The agent, in some embodiments, is a nucleic acid that encodes asarcoma-associated antigen operably linked to a promoter for expressingthe sarcoma-associated antigen. In other embodiments, the agent is apolypeptide comprising the sarcoma-associated antigen. In still otherembodiments, the agent is a host cell that expresses thesarcoma-associated antigen; preferably the host cell also expresses aMHC molecule. In yet other embodiments, the agent is a complex of apeptide derived from the sarcoma-associated antigen and a MHC molecule.

The composition also includes, in certain embodiments, an adjuvant orcytokine and/or one or more cytotoxic or chemotherapeutic agents. Thecompositions optionally includes a pharmaceutically acceptable carrier.

In another aspect of the invention, compositions are provided thatinclude an agent that selectively binds to a sarcoma-associated antigenor a nucleic acid molecule that encodes it. The nucleic acid moleculeincludes a nucleotide sequence selected from the group consisting of:(a) an isolated nucleic acid molecule comprising a nucleotide sequencethat is at least 90% identical to the nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 3, 5-8, 10-13, 99, 102 and 104 and(b) nucleic acid molecules that differ from the nucleic acid moleculesof (a) in codon sequence due to the degeneracy of the genetic code. Insome embodiments the nucleic acid molecule includes a nucleotidesequence selected from the group consisting of: (a) an isolated nucleicacid molecule comprising a nucleotide sequence that is at least 90%identical to the nucleotide sequence selected from the group consistingof SEQ ID NOs: 5-7, 10-13, 102 and 104 and (b) nucleic acid moleculesthat differ from the nucleic acid molecules of (a) in codon sequence dueto the degeneracy of the genetic code. In some embodiments the nucleicacid molecule includes a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 102 and 104; in other embodiments thenucleic acid molecule includes the nucleotide sequence set forth as SEQID NO: 10. In other embodiments the nucleic acid molecule includes thenucleotide sequence set forth as SEQ ID NO: 121. In still otherembodiments the nucleic acid molecule includes the nucleotide sequenceset forth in SEQ ID NO: 123. In yet other embodiments the nucleic acidmolecule includes the nucleotide sequence set forth in SEQ ID NO: 125.In still other embodiments the nucleic acid molecule includes thenucleotide sequence set forth in SEQ ID NOs: 127, 129 or 131. In otherembodiments, the sarcoma-associated antigen includes the amino acidsequence set forth as SEQ ID NO: 55, 56, 114 or 116 or a fragmentthereof that is at least eight amino acids in length. In someembodiments the sarcoma-associated antigen includes the amino acidsequence set forth as SEQ ID NO: 55. In certain embodiments thesarcoma-associated antigen includes the amino acid sequence set forth asSEQ ID NO: 122. In still other embodiments the sarcoma-associatedantigen includes the amino acid sequence set forth as SEQ ID NO: 124. Inyet other embodiments the sarcoma-associated antigen includes the aminoacid sequence set forth as SEQ ID NO: 126. In still other embodimentsthe sarcoma-associated antigen includes the amino acid sequence setforth as SEQ ID NOs: 128, 130 or 132. The agents in this aspect of theinvention include nucleic acids and polypeptides, preferably antibodiesor antigen-binding fragments thereof. Preferred antibodies includemonoclonal antibodies (particularly chimeric, human, or humanizedantibodies), and single chain antibodies; preferred antibody fragmentsinclude F(ab′)₂, Fab, Fd, or Fv fragments.

In certain embodiments, the antibody or antigen-binding fragment isconjugated to cytotoxic or chemotherapeutic agent. In other embodiments,the composition includes one or more cytotoxic or chemotherapeuticagent. In still other embodiments, the composition includes apharmaceutically acceptable carrier.

The use of the nucleotide and amino acid sequence as set forth as SEQ IDNOs: 133 and 134, respectively, in any of the compositions and methodsdescribed herein are also provided.

The invention also involves the use of the genes, gene products,fragments thereof, agents which bind thereto, and other compositions andmolecules described herein in the preparation of medicaments. Aparticular medicament is for treating cancer.

These and other aspects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides the mRNA expression patterns of serologically definedsarcoma antigens. FIG. 1A shows the results of the RT-PCR analysis ofNY-SAR-12, -35, and -41 in a panel of 17 normal tissues (Lanes 1, brain;2, kidney; 3, liver; 4, pancreas; 5, placenta; 6, testis; 7, fetalbrain; 8, small intestine; 9, heart; 10, prostate; 11, adrenal gland;12, spleen; 13, colon; 14, stomach; 15, lung; 16, bladder; and 17,ovary). FIG. 1B provides the results of the quantitative real-timeRT-PCR analysis of NY-SAR-35 in various normal tissues. FIG. 1C showsthe results of the RT-PCR analysis of NY-SAR-35 expression in sarcomacell lines and sarcoma tissue (Lane 1, fibrosarcoma; 2,rhabdomyosarcoma; 3, leiomyosarcoma; and 4, normal testis). FIG. 1Dprovides the results of the Northern blot analysis of NY-SAR-35 invarious normal tissues (Lane 1, spleen; 2, thymus; 3, prostate; 4,testis; 5, ovary; 6, small intestine; 7, colon mucosa; and 8, peripheralblood leukocytes).

FIG. 2 provides the nucleotide and predicted amino acid sequence ofNY-SAR-35 from each of the four ATG codons. The underlined lettersindicate the signal peptide and the italicized letters indicate thetransmembrane domain. The letters shown in gray represent the trefoildomain, while the letters that are underlined and italicized representthe other hydrophilic turn.

FIG. 3 provides the results of Western blot assay of recombinantNY-SAR-35 proteins in E. coli. Three colonies of each domain clonedplasmid were picked and cultured by IPTG induction. After a four hourinduction, total proteins from each of the colonies were separated bySDS-gel electrophoresis. The protein gel was immunoblotted on a membranewith a His-epitope monoclonal antibody. Lanes 1, 2, and 3-whole protein(from the first ATG codon); Lanes 4, 5 and 6-MH7 protein; Lanes 7, 8,and 9-extracellular protein and Lanes 10 and 11-E. coli lysate asnegative control.

FIG. 4 provides the real-time RT-PCR analysis of NY-SAR-35 mRNA invarious normal tissues and non-small cell lung cancer specimens.NY-SAR-35 was expressed in normal testis (83.2 ag) at a level thatwas >1,000 times the level detected in all other normal tissues. In 2 of9 cases of non-small cell lung cancer examined, the level of NY-SAR-35expression was equivalent to 0.15 (12.5 ag) and 0.13 (10.8 ag) times thelevel detected in normal testis, or approximately 100 times the leveldetected in normal tissues.

DETAILED DESCRIPTION OF THE INVENTION

The screening of cDNA expression libraries derived from human tumorswith autologous antibody (SEREX) has proven to be a powerful method fordefining the structure of tumor antigens recognized by the humoralimmune system, and has led to the identification of new targets forcancer immunotherapy. The current study examined the humoral immuneresponse of sarcoma patients to CT antigens. Sera from patients whichshowed a humoral immune response to CT antigens were subsequently usedto screen cDNA libraries derived from CT-rich sarcoma cell lines,leading to the identification of antigens not before associated withcancer along with several novel antigens associated with asarcoma-related immune response, including a novel CT antigen,NY-SAR-35.

Sarcoma-associated antigens were identified with an optimized SEREXanalysis method. Cell lines that were rich in CT antigen expression werechosen as the source of cDNA. Additionally, sera was obtained from agroup of patients that were actively mounting a humoral immune responseto a panel of known CT antigens. This optimized SEREX analysis led tothe identification of 113 antigens reactive with serum IgG of sarcomapatients. The antigens identified were further evaluated forcancer-restricted expression and the frequency of eliciting antibodyresponses in normal individuals as well as cancer patients.

In the first round of immunoscreenings, twenty-four of 72 antigens (33%)were found to have a serological profile that was not restricted tocancer patients, as evidenced by their reactivity with normal sera,while 48 antigens had a cancer-related serological profile, reactingonly with sera from cancer patients. Notable antigens belonging to thislatter category include the CT antigens, NY-SAR-36/SSX-1,NY-SAR-43/SSX-4 and NY-SAR-35. Although the antibody response in thesestudies to NY-SAR-4/FH was most frequent, occurring in 5/39 (13%)sarcoma patients, no individual antigen was serodominant. NY-SAR-4 isequivalent to fumarate hydratase (FH), an enzyme of the tricarboxylicacid cycle. This serological response to NY-SAR-4/FH may be of interestgiven the recent finding that germ line mutations in the FH gene areassociated with a predisposition to uterine and cutaneous leiomyomata,and also renal cell carcinoma (Tomlinson I P, et al. Germline mutationsin FH predispose to dominantly inherited uterine fibroids, skinleiomyomata and papillary renal cell cancer. Nat Genet. 2002 Apr.;30(4):406-10).

In addition, 6 tissue-restricted antigens, LAGE-1/NY-SAR-17,SSX1/NY-SAR-36, SSX4/NY-SAR43, NESG1/NY-SAR-12, NY-SAR-35, and NY-SAR-41were identified. Two of these antigens, NY-SAR-35, and NY-SAR-41 arenovel gene products, and a third, NESG1/NY-SAR-12 (Li Z, Yao K, Cao Y.Molecular cloning of a novel tissue-specific gene from humannasopharyngeal epithelium. Gene 1999 Sep. 3; 237(1):235-40), has notbeen previously studied in relation to cancer. NY-SAR-35 furtherrepresents a newly defined CT antigen expressed exclusively in normaltestis, melanoma, sarcoma, lung cancer and breast cancer.

The second round of immunoscreenings performed led to the identificationof 41 additional SEREX-defined sarcoma antigens, 11 of which are novelgene products (NY-SAR-77, -79, -80, -84, -88, -92, -95, -97, -104, 105and -113). Within this group of 41 sarcoma antigens are three knowntestis-restricted antigens (NY-SAR-78/TSP-NY, NY-SAR-89/SSX2 andNY-SAR-99/SSX3), two differentially expressed antigens that are novelgene products (NY-SAR-92 and NY-SAR-97) and a tissue-restricted antigenthat has not been previously studied in relation to cancer(NY-SAR-96/MCSP).

Table 1, below, provides a list of the sarcoma-associated antigens andtheir corresponding sequence identification numbers. The antigens listedinclude those that were found to be uncharacterized gene products aswell as those sarcoma-associated antigens that exhibitedcancer-restricted expression and were not found in the SEREX Database.

TABLE 1 Sarcoma-Associated Antigens (Uncharacterized Gene Products andCancer-Related Antigens not Found in the SEREX Database) SequenceIdentification NY- Number (nucleotide and SAR- amino acid sequence,Antigen respectively) 3 SEQ ID NOs: 1 and 46 10 SEQ ID NOs: 2 and 47 16SEQ ID NOs: 3 and 48 22 SEQ ID NOs: 4 and 49 23 SEQ ID NOs: 5 and 50 24SEQ ID NOs: 6 and 51 27 SEQ ID NOs: 7 and 52 28 SEQ ID NOs: 8 and 53 29SEQ ID NOs: 9 and 54 35 SEQ ID NOs: 10 and 55 41 SEQ ID NOs: 11 and 5648 SEQ ID NOs: 12 and 57 62 SEQ ID NOs: 13 and 58 71 SEQ ID NOs: 14 and59 12 SEQ ID NOs: 15 and 60 4 SEQ ID NOs: 16 and 61 5 SEQ ID NOs: 17 and62 8 SEQ ID NOs: 18 and 63 9 SEQ ID NOs: 19 and 64 20 SEQ ID NOs: 20 and65 21 SEQ ID NOs: 21 and 66 25 SEQ ID NOs: 22 and 67 26 SEQ ID NOs: 23and 68 30 SEQ ID NOs: 24 and 69 34 SEQ ID NOs: 25 and 70 36 SEQ ID NOs:26 and 71 37 SEQ ID NOs: 27 and 72 38 SEQ ID NOs: 28 and 73 39 SEQ IDNOs: 29 and 74 40 SEQ ID NOs: 30 and 75 42 SEQ ID NOs: 31 and 76 43 SEQID NOs: 32 and 77 46 SEQ ID NOs: 33 and 78 49 SEQ ID NOs: 34 and 79 50SEQ ID NOs: 35 and 80 51 SEQ ID NOs: 36 and 81 52 SEQ ID NOs: 37 and 8256 SEQ ID NOs: 38 and 83 57 SEQ ID NOs: 39 and 84 59 SEQ ID NOs: 40 and85 60 SEQ ID NOs: 41 and 86 63 SEQ ID NOs: 42 and 87 67 SEQ ID NOs: 43and 88 69 SEQ ID NOs: 44 and 89 70 SEQ ID NOs: 45 and 90 77 SEQ ID NOs:97 and 109 79 SEQ ID NOs: 98 and 110 80 SEQ ID NOs: 99 and 111 84 SEQ IDNOs: 100 and 112 88 SEQ ID NOs: 101 and 113 92 SEQ ID NOs: 102 and 11495 SEQ ID NOs: 103 and 115 97 SEQ ID NOs: 104 and 116 104 SEQ ID NOs:105 and 117 105 SEQ ID NOs: 106 and 118 113 SEQ ID NOs: 107 and 119 96SEQ ID NOs: 108 and 120

The invention relates, in part, to the sarcoma-associated antigensdefined herein and the nucleic acid molecules that encode them. Theinvention further relates to the use of the nucleic acid molecules,polypeptides and fragments thereof associated with sarcoma in methodsand compositions for the diagnosis and treatment of diseases, such ascancer.

As used herein, the term “sarcoma-associated antigens” meanspolypeptides that elicit specific immune responses to the polypeptidewhen expressed by a tumor cell and thus, include sarcoma-associatedpolypeptides (including proteins) and fragments of sarcoma-associatedpolypeptides, that are recognized by the immune system (e.g., byantibodies and/or T lymphocytes). In part, the invention relates tosarcoma-associated antigens as well as the nucleic acid molecules thatencode the sarcoma-associated antigens. As used herein, the “nucleicacid molecules that encode” means the nucleic acid molecules that codefor the immunogenic sarcoma-associated polypeptides or immunogenicfragments thereof. These nucleic acid molecules may be DNA or may be RNA(e.g. mRNA). The sarcoma-associated nucleic acid molecules of theinvention also encompass variants of the nucleic acid moleculesdescribed herein. These variants may be splice variants or allelicvariants of certain sequences provided. Variants of the nucleic acidmolecules of the invention are intended to include homologs and alleleswhich are described further below. Further, as used herein, the term“sarcoma-associated molecules” includes sarcoma-associated antigens(polypeptides and fragments thereof) as well as sarcoma-associatednucleic acids. In all embodiments, human sarcoma-associated antigens andthe encoding nucleic acid molecules thereof, are preferred.

In one aspect, the invention provides isolated nucleic acid moleculesthat encode the sarcoma-associated antigens defined herein. The isolatednucleic acid molecules of this aspect of the invention comprise: (a)nucleotide sequences selected from the group consisting of nucleotidesequences set forth as SEQ ID NOs: 1-14 and 97-107 (b) isolated nucleicacid molecules which hybridize under highly stringent conditions to thenucleic acid molecules of (a) and which code for a sarcoma-associatedantigen, (c) nucleic acid molecules that differ from (a) or (b) due tothe degeneracy of the genetic code, and (d) complements of (a), (b) or(c).

As used herein the term “isolated nucleic acid molecule” means: (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

The sarcoma-associated nucleic acid molecules of the invention alsointended to encompass homologs and alleles which can be identified byconventional techniques. Identification of human and other organismhomologs of sarcoma-associated polypeptides will be familiar to those ofskill in the art. In general, nucleic acid hybridization is a suitablemethod for identification of homologous sequences of another species(e.g., human, cow, sheep), which correspond to a known sequence.Standard nucleic acid hybridization procedures can be used to identifyrelated nucleic acid sequences of selected percent identity. Forexample, one can construct a library of cDNAs reverse transcribed fromthe mRNA of a selected tissue and use the nucleic acids that encodesarcoma-associated antigens identified herein to screen the library forrelated nucleotide sequences. The screening preferably is performedusing high-stringency conditions to identify those sequences that areclosely related by sequence identity. Nucleic acids so identified can betranslated into polypeptides and the polypeptides can be tested foractivity.

The term “high stringency” as used herein refers to parameters withwhich the art is familiar. Nucleic acid hybridization parameters may befound in references that compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, high-stringencyconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4(pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDSis sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed, for example, in 2×SSC at room temperature and then at0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C.

There are other conditions, reagents, and so forth that can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of the sarcoma-associated nucleic acids of theinvention (e.g., by using lower stringency conditions). The skilledartisan also is familiar with the methodology for screening cells andlibraries for expression of such molecules, which then are routinelyisolated, followed by isolation of the pertinent nucleic acid moleculeand sequencing.

In general, homologs and alleles typically will share at least 90%nucleotide identity and/or at least 95% amino acid identity to thesequences of sarcoma-associated nucleic acids and polypeptides,respectively, in some instances will share at least 95% nucleotideidentity and/or at least 97% amino acid identity, in other instanceswill share at least 97% nucleotide identity and/or at least 98% aminoacid identity, in other instances will share at least 99% nucleotideidentity and/or at least 99% amino acid identity, and in other instanceswill share at least 99.5% nucleotide identity and/or at least 99.5%amino acid identity. The homology can be calculated using various,publicly available software tools developed by NCBI (Bethesda, Md.) thatcan be obtained through the internet. Exemplary tools include the BLASTsystem available from the website of the National Center forBiotechnology Information (NCBI) at the National Institutes of Health.Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well asKyte-Doolittle hydropathic analysis can be obtained using the MacVectorsequence analysis software (Oxford Molecular Group). Watson-Crickcomplements of the foregoing nucleic acids also are embraced by theinvention.

In another aspect of the invention, unique fragments are provided whichinclude unique fragments of the nucleotide sequences of the inventionand complements thereof. The invention, in a preferred embodiment,provides unique fragments of SEQ ID NO: 10, 11, 15, 102, 104 or 108 andcomplements thereof. In another preferred embodiment, provides uniquefragments of SEQ ID NO: 10 and complements thereof. In other embodimentsthe unique fragment includes the nucleotide sequence set forth as SEQ IDNO: 121. In still other embodiments the unique fragments includes thesequence set forth as SEQ ID NO: 123, 125, 127, 129 or 131. A uniquefragment is one that is a ‘signature’ for the larger nucleic acid. It,for example, is long enough to assure that its precise sequence is notfound in molecules outside of the nucleic acid molecules that encode thesarcoma-associated antigens defined above. Those of ordinary skill inthe art may apply no more than routine procedures to determine if afragment is unique within the human genome. In some instances the uniquefragment is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 amino acids inlength.

Unique fragments can be used as probes in Southern blot assays toidentify such nucleic acid molecules, or can be used as probes inamplification assays such as those employing the polymerase chainreaction (PCR), including, but not limited to RT-PCR and RT-real-timePCR. As known to those skilled in the art, large probes such as 200nucleotides or more are preferred for certain uses such as Southernblots, while smaller fragments will be preferred for uses such as PCR.Unique fragments also can be used to produce fusion proteins forgenerating antibodies or determining binding of the polypeptidefragments, or for generating immunoassay components. Likewise, uniquefragments can be employed to produce nonfused fragments of thesarcoma-associated polypeptides useful, for example, in the preparationof antibodies and in immunoassays.

In screening for sarcoma-associated antigen genes, a Southern blot maybe performed using the foregoing conditions, together with a detectablylabeled probe (e.g. radioactive or chemiluminescent probes). Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film or analyzed using aphosphorimager device to detect the radioactive or chemiluminescentsignal. In screening for the expression of sarcoma-associated antigennucleic acids, Northern blot hybridizations using the foregoingconditions can be performed on samples taken from cancer patients orsubjects suspected of having a condition characterized by abnormal cellproliferation or neoplasia. Amplification protocols such as polymerasechain reaction using primers that hybridize to the sequences presentedalso can be used for detection of the sarcoma-associated antigen genesor expression thereof.

Identification of related sequences can also be achieved usingpolymerase chain reaction (PCR) and other amplification techniquessuitable for cloning related nucleic acid sequences. Preferably, PCRprimers are selected to amplify portions of a nucleic acid sequencebelieved to be conserved (e.g., a catalytic domain, a DNA-bindingdomain, etc.). Again, nucleic acids are preferably amplified from atissue-specific library (e.g., testis). One also can use expressioncloning utilizing the antisera described herein to identify nucleicacids that encode related antigenic proteins in humans or other speciesusing the SEREX procedure to screen the appropriate expressionlibraries. (See: Sahin et al. Proc. Natl. Acad. Sci. USA 92:11810-11813,1995).

The invention also includes degenerate nucleic acids that includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongatingsarcoma-associated polypeptide. Similarly, nucleotide sequence tripletswhich encode other amino acid residues include, but are not limited to:CCA, CCC, CCG, and CCT (proline codons); CGA, CGC, CGG, CGT, AGA, andAGG (arginine codons); ACA, ACC, ACG, and ACT (threonine codons); AACand AAT (asparagine codons); and ATA, ATC, and ATT (isoleucine codons).Other amino acid residues may be encoded similarly by multiplenucleotide sequences. Thus, the invention embraces degenerate nucleicacids that differ from the biologically isolated nucleic acids in codonsequence due to the degeneracy of the genetic code.

The invention also provides modified nucleic acid molecules, whichinclude additions, substitutions and deletions of one or morenucleotides (preferably 1-20 nucleotides). In preferred embodiments,these modified nucleic acid molecules and/or the polypeptides theyencode retain at least one activity or function of the unmodifiednucleic acid molecule and/or the polypeptides, such as antigenicity,receptor binding, etc. In certain embodiments, the modified nucleic acidmolecules encode modified polypeptides, preferably polypeptides havingconservative amino acid substitutions as are described elsewhere herein.The modified nucleic acid molecules are structurally related to theunmodified nucleic acid molecules and in preferred embodiments aresufficiently structurally related to the unmodified nucleic acidmolecules so that the modified and unmodified nucleic acid moleculeshybridize under stringent conditions known to one of skill in the art.

For example, modified nucleic acid molecules that encode polypeptideshaving single amino acid changes can be prepared. Each of these nucleicacid molecules can have one, two or three nucleotide substitutionsexclusive of nucleotide changes corresponding to the degeneracy of thegenetic code as described herein. Likewise, modified nucleic acidmolecules that encode polypeptides having two amino acid changes can beprepared which have, e.g., 2-6 nucleotide changes. Numerous modifiednucleic acid molecules like these will be readily envisioned by one ofskill in the art, including for example, substitutions of nucleotides incodons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and soon. In the foregoing example, each combination of two amino acids isincluded in the set of modified nucleic acid molecules, as well as allnucleotide substitutions which code for the amino acid substitutions.Additional nucleic acid molecules that encode polypeptides havingadditional substitutions (i.e., 3 or more), additions or deletions(e.g., by introduction of a stop codon or a splice site(s)) also can beprepared and are embraced by the invention as readily envisioned by oneof ordinary skill in the art. Any of the foregoing nucleic acids orpolypeptides can be tested by routine experimentation for retention ofactivity or structural relation to the nucleic acids and/or polypeptidesdisclosed herein. As used herein the terms: “deletion”, “addition”, and“substitution” mean deletion, addition, and substitution changes toabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleic acids of asequence of the invention.

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the invention,preferably operably linked to a promoter is provided. In a relatedaspect, host cells transformed or transfected with such expressionvectors also are provided. As used herein, a “vector” may be any of anumber of nucleic acid molecules into which a desired sequence may beinserted by restriction and ligation for transport between differentgenetic environments or for expression in a host cell. Vectors aretypically composed of DNA although RNA vectors are also available.Vectors include, but are not limited to, plasmids, phagemids, and virusgenomes. A cloning vector is one which is able to replicate in a hostcell, and which is further characterized by one or more endonucleaserestriction sites at which the vector may be cut in a determinablefashion and into which a desired DNA sequence may be ligated such thatthe new recombinant vector retains its ability to replicate in the hostcell. In the case of plasmids, replication of the desired sequence mayoccur many times as the plasmid increases in copy number within the hostbacterium or just a single time per host before the host reproduces bymitosis. In the case of phage, replication may occur actively during alytic phase or passively during a lysogenic phase. An expression vectoris one into which a desired DNA sequence may be inserted by restrictionand ligation such that it is operably joined to regulatory sequences andmay be expressed as an RNA transcript. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellswhich have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art, e.g., -galactosidase or alkalinephosphatase, and genes which visibly affect the phenotype of transformedor transfected cells, hosts, colonies or plaques, e.g., greenfluorescent protein. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. As used herein,“operably joined” and “operably linked” are used interchangeably andshould be construed to have the same meaning. If it is desired that thecoding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region is operably joined to a coding sequence if thepromoter region is capable of effecting transcription of that DNAsequence such that the resulting transcript can be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Often, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

It will also be recognized that the invention embraces the use of thesarcoma-associated nucleic acid molecules and genomic sequences inexpression vectors, as well as to transfect host cells and cell lines,be these prokaryotic, e.g., E. coli, or eukaryotic, e.g., CHO cells, COScells, yeast expression systems, and recombinant baculovirus expressionin insect cells. Especially useful are mammalian cells such as human,mouse, hamster, pig, goat, primate, etc. They may be of a wide varietyof tissue types, including mast cells, fibroblasts, oocytes, andlymphocytes, and may be primary cells and cell lines. Specific examplesinclude dendritic cells, peripheral blood leukocytes, bone marrow stemcells and embryonic stem cells. The expression vectors require that thepertinent sequence, i.e., those nucleic acids described supra, beoperably linked to a promoter.

The invention, in one aspect, also permits the construction ofsarcoma-associated antigen gene “knock-outs” and “knock-ins” in cellsand in animals, providing materials for studying certain aspects ofcancer and immune system responses to cancer.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA or RNA encoding a sarcoma-associated antigen, a mutantsarcoma-associated antigen, fragments, or variants thereof. Theheterologous DNA or RNA is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pcDNA1.1 and pCDM8 (Invitrogen) that contain a selectable marker(which facilitates the selection of stably transfected cell lines) andcontain the human cytomegalovirus (CMV) enhancer-promoter sequences.Additionally, suitable for expression in primate or canine cell lines isthe pCEP4 vector (Invitrogen), which contains an Epstein Barr virus(EBV) origin of replication, facilitating the maintenance of plasmid asa multicopy extrachromosomal element. Another expression vector is thepEF-BOS plasmid containing the promoter of polypeptide Elongation Factor1, which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mizushima and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdescribed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996).

The invention also embraces kits termed expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also includes kits for amplification of asarcoma-associated antigen nucleic acid, including at least one pair ofamplification primers which hybridize to a sarcoma-associated nucleicacid. The primers preferably are about 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides inlength and are non-overlapping to prevent formation of “primer-dimers”.One of the primers will hybridize to one strand of thesarcoma-associated nucleic acid and the second primer will hybridize tothe complementary strand of the sarcoma-associated nucleic acid, in anarrangement which permits amplification of the sarcoma-associatednucleic acid. Selection of appropriate primer pairs is standard in theart. For example, the selection can be made with assistance of acomputer program designed for such a purpose, optionally followed bytesting the primers for amplification specificity and efficiency.

The invention, in another aspect provides isolated polypeptides(including whole proteins and partial proteins) encoded by the foregoingsarcoma-associated nucleic acids. Examples of the amino acid sequencesencoded by the foregoing sarcoma-associated nucleic acids are set forthas SEQ ID NOs: 46-90 and 109-120. The amino acids of the invention arealso intended to encompass amino acid sequences that result from thetranslation of the nucleic acid sequences provided herein in a differentreading frame. In one preferred embodiment of the invention apolypeptide is provided which comprises the polypeptide sequence setforth as SEQ ID NO: 55, 56, 60, 114, 116 or 120. In another preferredembodiment a polypeptide is provided which comprises the polypeptidesequence set forth as SEQ ID NO: 122. In still another preferredembodiment a polypeptide is provided which comprises the polypeptidesequence set forth as SEQ ID NO: 124. In still other embodimentspolypeptides are provided which comprise the polypeptide sequence setforth as SEQ ID NO: 126, 128, 130 or 132. Such polypeptides are useful,for example, alone or as fusion proteins to generate antibodies, and ascomponents of an immunoassay or diagnostic assay. Immunogenicsarcoma-associated polypeptides can be isolated from biological samplesincluding tissue or cell homogenates, and can also be expressedrecombinantly in a variety of prokaryotic and eukaryotic expressionsystems by constructing an expression vector appropriate to theexpression system, introducing the expression vector into the expressionsystem, and isolating the recombinantly expressed protein. Fragments ofthe immunogenic sarcoma-associated polypeptides (including immunogenicpeptides) also can be synthesized chemically using well-establishedmethods of peptide synthesis. Thus, fragments of the disclosedpolypeptides are useful for eliciting an immune response. In oneembodiment fragments of a polypeptide which comprises SEQ ID NO: 55, 56,60, 114, 116 or 120 that are at least eight amino acids in length andexhibit immunogenicity are provided. In one embodiment fragments of apolypeptide which comprises SEQ ID NO: 55 that are at least eight aminoacids in length and exhibit immunogenicity are provided. In anotherembodiment a polypeptide is provided which comprises the polypeptidesequence set forth as SEQ ID NO: 122. In still another preferredembodiments a polypeptide is provided which comprises the polypeptidesequence set forth as SEQ ID NO: 124. In still other embodimentspolypeptides are provided which comprise the polypeptide sequence setforth as SEQ ID NO: 126, 128, 130 or 132.

Fragments of a polypeptide preferably are those fragments that retain adistinct functional capability of the polypeptide. Functionalcapabilities that can be retained in a fragment of a polypeptide includeinteraction with antibodies or MHC molecules (e.g. immunogenicfragments), interaction with other polypeptides or fragments thereof,selective binding of nucleic acids or proteins, and enzymatic activity.One important activity is the ability to provoke in a subject an immuneresponse. As will be recognized by those skilled in the art, the size ofthe fragment that can be used for inducing an immune response willdepend upon factors such as whether the epitope recognized by anantibody is a linear epitope or a conformational epitope or theparticular MHC molecule that binds to and presents the fragment (e.g.HLA class I or II). Thus, some immunogenic fragments ofsarcoma-associated polypeptides will consist of longer segments whileothers will consist of shorter segments, (e.g. about 5, 6, 7, 8, 9, 10,11 or 12 or more amino acids long, including each integer up to the fulllength of the sarcoma-associated polypeptide). Those skilled in the artare well versed in methods for selecting immunogenic fragments ofpolypeptides.

The invention embraces variants of the sarcoma-associated polypeptidesdescribed above. As used herein, a “variant” of a sarcoma-associatedantigen polypeptide is a polypeptide which contains one or moremodifications to the primary amino acid sequence of a sarcoma-associatedpolypeptide. Modifications which create a sarcoma-associated antigenvariant can be made to a sarcoma-associated polypeptide 1) to reduce oreliminate an activity of a sarcoma-associated polypeptide; 2) to enhancea property of a sarcoma-associated polypeptide, such as proteinstability in an expression system or the stability of protein-proteinbinding; 3) to provide a novel activity or property to asarcoma-associated polypeptide, such as addition of an antigenic epitopeor addition of a detectable moiety; or 4) to provide equivalent orbetter binding to a MHC molecule.

Modifications to a sarcoma-associated polypeptide are typically made tothe nucleic acid which encodes the sarcoma-associated polypeptide, andcan include deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids or non-amino acid moieties.Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the sarcoma-associated antigen amino acid sequence. One of skill inthe art will be familiar with methods for predicting the effect onprotein conformation of a change in protein sequence, and can thus“design” a variant sarcoma-associated polypeptide according to knownmethods. One example of such a method is described by Dahiyat and Mayoin Science 278:82-87, 1997, whereby proteins can be designed de novo.The method can be applied to a known protein to vary a only a portion ofthe polypeptide sequence. By applying the computational methods ofDahiyat and Mayo, specific variants of a sarcoma-associated polypeptidecan be proposed and tested to determine whether the variant retains adesired conformation.

In general, variants include sarcoma-associated polypeptides which aremodified specifically to alter a feature of the polypeptide unrelated toits desired physiological activity. For example, cysteine residues canbe substituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of asarcoma-associated polypeptide by eliminating proteolysis by proteasesin an expression system (e.g., dibasic amino acid residues in yeastexpression systems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a sarcoma-associatedpolypeptide preferably preserve the amino acid reading frame of thecoding sequence, and preferably do not create regions in the nucleicacid which are likely to hybridize to form secondary structures, such ahairpins or loops, which can be deleterious to expression of the variantpolypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant sarcoma-associated polypeptides) whichare silent as to the amino acid sequence of the polypeptide, but whichprovide preferred codons for translation in a particular host. Thepreferred codons for translation of a nucleic acid in, e.g., E. coli,are well known to those of ordinary skill in the art. Still othermutations can be made to the noncoding sequences of a sarcoma-associatedantigen gene or cDNA clone to enhance expression of the polypeptide. Theactivity of variants of sarcoma-associated polypeptides can be tested bycloning the gene encoding the variant sarcoma-associated polypeptideinto a bacterial or mammalian expression vector, introducing the vectorinto an appropriate host cell, expressing the variant sarcoma-associatedpolypeptide, and testing for a functional capability of thesarcoma-associated polypeptides as disclosed herein. For example, thevariant sarcoma-associated polypeptide can be tested for reaction withautologous or allogeneic sera as described in the Examples. Preparationof other variant polypeptides may favor testing of other activities, aswill be known to one of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in immunogenic sarcoma-associated polypeptidesto provide functionally equivalent variants, or homologs of theforegoing polypeptides, i.e., the variants retain the functionalcapabilities of the immunogenic sarcoma-associated polypeptides. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references that compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Exemplary functionally equivalentvariants or homologs of the sarcoma-associated polypeptides includeconservative amino acid substitutions of in the amino acid sequences ofproteins disclosed herein. Conservative substitutions of amino acidsinclude substitutions made amongst amino acids within the followinggroups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;(f) Q, N; and (g) E, D. Therefore, one can make conservative amino acidsubstitutions to the amino acid sequence of the sarcoma-associatedantigens disclosed herein and retain the specific antibody-bindingcharacteristics of the antigens.

Likewise, upon determining that a peptide derived from asarcoma-associated polypeptide is presented by an MHC molecule andrecognized by antibodies or T lymphocytes (e.g., helper T cells orCTLs), one can make conservative amino acid substitutions to the aminoacid sequence of the peptide, particularly at residues which are thoughtnot to be direct contact points with the MHC molecule. For example,methods for identifying functional variants of HLA class II bindingpeptides are provided in a published PCT application of Strominger andWucherpfennig (PCT/US96/03182). Peptides bearing one or more amino acidsubstitutions also can be tested for concordance with known HLA/MHCmotifs prior to synthesis using, e.g. the computer program described byD'Amaro and Drijfhout (D'Amaro et al., Human Immunol. 43:13-18, 1995;Drijfhout et al., Human Immunol. 43:1-12, 1995). The substitutedpeptides can then be tested for binding to the MHC molecule andrecognition by antibodies or T lymphocytes when bound to MHC. Thesevariants can be tested for improved stability and are useful, interalia, in vaccine compositions.

Conservative amino-acid substitutions in the amino acid sequence ofsarcoma-associated polypeptides to produce functionally equivalentvariants of sarcoma-associated polypeptides typically are made byalteration of a nucleic acid encoding a sarcoma-associated polypeptide.Such substitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a sarcoma-associatedpolypeptide. Where amino acid substitutions are made to a small uniquefragment of a sarcoma-associated polypeptide, such as an antigenicepitope recognized by autologous or allogeneic sera or T lymphocytes,the substitutions can be made by directly synthesizing the peptide. Theactivity of functionally equivalent variants of sarcoma-associatedpolypeptides can be tested by cloning the gene encoding the alteredsarcoma-associated polypeptide into a bacterial or mammalian expressionvector, introducing the vector into an appropriate host cell, expressingthe altered polypeptide, and testing for a functional capability of thesarcoma-associated polypeptides as disclosed herein. Peptides that arechemically synthesized can be tested directly for function, e.g., forbinding to antisera recognizing associated antigens.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. In one aspect of the invention a methodof identifying cancer-associated antigens is provided. Novelcancer-associated antigens can be identified by obtaining a biologicalsample from a subject, determining the reactivity of the biologicalsample with one or more known cancer-associated antigens, andsubsequently using the reactive biological sample to screen anexpression library to identify novel cancer-associated antigens as wellas proteins previously known but not previously associated with cancer.

As used herein, a “subject” is preferably a human, non-human primate,cow, horse, pig, sheep, goat, dog, cat or rodent. In all embodiments,human subjects are preferred. In some embodiments, the subject issuspected of having cancer or has been diagnosed with cancer. Cancers inwhich the sarcoma-associated nucleic acid or polypeptide aredifferentially expressed include sarcoma.

As used herein, a biological sample includes, but is not limited to:tissue, cells, or body fluid (e.g. serum, blood, lymph node fluid,etc.). The fluid sample may include cells and/or fluid. The tissue andcells may be obtained from a subject or may be grown in culture (e.g.from a cell line). As used herein, a biological sample is body fluid,tissue or cells obtained from a subject using methods well-known tothose of ordinary skill in the related medical arts.

The invention in another aspect permits the isolation of thecancer-associated antigens described herein. A variety of methodologieswell-known to the skilled practitioner can be utilized to obtainisolated cancer-associated antigens. The proteins may be purified fromcells which naturally produce the protein by chromatographic means orimmunological recognition. Alternatively, an expression vector may beintroduced into cells to cause production of the protein. In anothermethod, mRNA transcripts may be microinjected or otherwise introducedinto cells to cause production of the encoded protein. Translation ofmRNA in cell-free extracts such as the reticulocyte lysate system alsomay be used to produce the protein. Those skilled in the art also canreadily follow known methods for isolating cancer-associated antigens.These include, but are not limited to, chromatographic techniques suchas immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography, and immune-affinity chromatography.

The invention also involves diagnosing or monitoring cancer in subjectsby determining the presence of an immune response to one or moresarcoma-associated antigens of the invention. In preferred embodiments,this determination is performed by assaying a bodily fluid obtained fromthe subject, preferably serum, blood, or lymph node fluid for thepresence of antibodies against the sarcoma-associated antigens describedherein. This determination may also be performed by assaying a tissue orcells from the subject for the presence of one or moresarcoma-associated antigens (or nucleic acid molecules that encode theseantigens) described herein. In another embodiment, the presence ofantibodies against at least one additional cancer antigen is determinedfor diagnosis of cancer. The additional antigen may be asarcoma-associated antigen as described herein or may be some othercancer-associated antigen. This determination may also be performed byassaying a tissue or cells from the subject for the presence of thesarcoma-associated antigens described herein.

Measurement of the immune response against one of the sarcoma-associatedantigens over time by sequential determinations permits monitoring ofthe disease and/or the effects of a course of treatment. For example, asample, such as serum, blood, or lymph node fluid, may be obtained froma subject, tested for an immune response to one of thesarcoma-associated antigens, and at a second, subsequent time, anothersample, may be obtained from the subject and similarly tested. Theresults of the first and second (or subsequent) tests can be compared asa measure of the onset, regression or progression of cancer, or, ifcancer treatment was undertaken during the interval between obtainingthe samples, the effectiveness of the treatment may be evaluated bycomparing the results of the two tests. In preferred embodiments thesarcoma-associated antigens are bound to a substrate. In other preferredembodiments the immune response of the biological sample to thesarcoma-associated antigens is determined with ELISA. Other methods willbe apparent to one of skill in the art.

Diagnostic methods of the invention also involve determining theaberrant expression of one or more of the sarcoma-associated antigensdescribed herein or the nucleic acid molecules that encode them. Suchdeterminations can be carried out via any standard nucleic acid assay,including the polymerase chain reaction or assaying with hybridizationprobes, which may be labeled, or by assaying biological samples withbinding partners (e.g., antibodies) for sarcoma-associated antigens.

The diagnostic methods of the invention can be used to detect thepresence of a disorder associated with aberrant expression of asarcoma-associated molecule, as well as to assess the progression and/orregression of the disorder such as in response to treatment (e.g.,chemotherapy, radiation). According to this aspect of the invention, themethod for diagnosing a disorder characterized by aberrant expression ofa sarcoma-associated molecule involve: detecting expression of asarcoma-associated molecule in a first biological sample obtained from asubject, wherein differential expression of the sarcoma-associatedmolecule compared to a control sample indicates that the subject has adisorder characterized by aberrant expression of a sarcoma-associatedmolecule, such as cancer.

As used herein, “aberrant expression” of a sarcoma-associated antigen isintended to include any expression that is statistically significantfrom the expected amount of expression. For example, expression of asarcoma-associated molecule (i.e., the sarcoma-associated antigen or thenucleic acid molecules that encode it) in a tissue that is not expectedto express the sarcoma-associated molecule would be included in thedefinition of “aberrant expression”. Likewise, expression of thesarcoma-associated molecule that is determined to be expressed at asignificantly higher or lower level than expected is also included.Therefore, a determination of the level of expression of one or more ofthe sarcoma-associated antigens and/or the nucleic acids that encodethem is diagnostic of cancer if the level of expression is above abaseline level determined for that tissue type. The baseline level ofexpression can be determined using standard methods known to those ofskill in the art. Such methods include, for example, assaying a numberof histologically normal tissue samples from subjects that areclinically normal (i.e. do not have clinical signs of cancer in thattissue type) and determining the mean level of expression for thesamples.

The level of expression of the nucleic acid molecules of the inventionor the antigens they encode can indicate cancer in the tissue when thelevel of expression is significantly more in the tissue than in acontrol sample. In some embodiments, a level of expression in thetissues that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% more than thelevel of expression in the control tissue indicates cancer in thetissue.

As used herein the term “control” means predetermined values, and alsomeans samples of materials tested in parallel with the experimentalmaterials. Examples include samples from control populations or controlsamples generated through manufacture to be tested in parallel with theexperimental samples.

As used herein the term “control” includes positive and negativecontrols which may be a predetermined value that can take a variety offorms. The control(s) can be a single cut-off value, such as a median ormean, or can be established based upon comparative groups, such as ingroups having normal amounts of sarcoma-associated molecules of theinvention and groups having abnormal amounts of sarcoma-associatedmolecules of the invention. Another example of a comparative group is agroup having a particular disease, condition and/or symptoms and a groupwithout the disease, condition and/or symptoms. Another comparativegroup is a group with a family history of a particular disease and agroup without such a family history of the particular disease. Thepredetermined control value can be arranged, for example, where a testedpopulation is divided equally (or unequally) into groups, such as alow-risk group, a medium-risk group and a high-risk group or intoquadrants or quintiles, the lowest quadrant or quintile beingindividuals with the lowest risk or lowest expression levels of asarcoma-associated molecule of the invention that is up-regulated incancer and the highest quadrant or quintile being individuals with thehighest risk or highest expression levels of a sarcoma-associatedmolecule of the invention that is up-regulated in cancer.

The predetermined value of a control will depend upon the particularpopulation selected. For example, an apparently healthy population willhave a different “normal” sarcoma-associated molecule expression levelrange than will a population which is known to have a conditioncharacterized by aberrant expression of the sarcoma-associated molecule.Accordingly, the predetermined value selected may take into account thecategory in which an individual falls. Appropriate ranges and categoriescan be selected with no more than routine experimentation by those ofordinary skill in the art. Typically the control will be based onapparently healthy individuals in an appropriate age bracket. As usedherein, the term “increased expression” means a higher level ofexpression relative to a selected control.

The invention involves in some aspects diagnosing or monitoring cancerby determining the level of expression of one or more sarcoma-associatednucleic acid molecules and/or determining the level of expression of oneor more sarcoma-associated polypeptides they encode. In some importantembodiments, this determination is performed by assaying a tissue samplefrom a subject for the level of expression of one or moresarcoma-associated nucleic acid molecules or for the level of expressionof one or more sarcoma-associated polypeptides encoded by the nucleicacid molecules of the invention.

The expression of the molecules of the invention may be determined usingroutine methods known to those of ordinary skill in the art. Thesemethods include, but are not limited to: direct RNA amplification,reverse transcription of RNA to cDNA, real-time RT-PCR, amplification ofcDNA, hybridization, and immunologically based assay methods, whichinclude, but are not limited to immunohistochemistry, antibody sandwichcapture assay, ELISA, and enzyme-linked immunospot assay (EliSpotassay). For example, the determination of the presence of level ofnucleic acid molecules of the invention in a subject or tissue can becarried out via any standard nucleic acid determination assay, includingthe polymerase chain reaction, or assaying with labeled hybridizationprobes. Such hybridization methods include, but are not limited tomicroarray techniques.

These methods of determining the presence and/or level of the moleculesof the invention in cells and tissues may include use of labels tomonitor the presence of the molecules of the invention. Such labels mayinclude, but are not limited to radiolabels or chemiluminescent labels,which may be utilized to determine whether a molecule of the inventionis expressed in a cell or tissue, and to determine the level ofexpression in the cell or tissue. For example, a fluorescently labeledor radiolabeled antibody that selectively binds to a polypeptide of theinvention may be contacted with a tissue or cell to visualize thepolypeptide in vitro or in vivo. These and other in vitro and in vivoimaging methods for determining the presence of the nucleic acid andpolypeptide molecules of the invention are well known to those ofordinary skill in the art.

The invention, therefore, also involves the use of agents such aspolypeptides that bind to sarcoma-associated antigens. Such agents canbe used in methods of the invention including the diagnosis and/ortreatment of cancer. Such binding agents can be used, for example, inscreening assays to detect the presence or absence of sarcoma-associatedantigens and can be used in quantitative binding assays to determinelevels of expression in biological samples and cells. Such agents alsomay be used to inhibit the native activity of the sarcoma-associatedpolypeptides, for example, by binding to such polypeptides.

According to this aspect, the binding polypeptides bind to an isolatednucleic acid or protein of the invention, including unique fragmentsthereof. Preferably, the binding polypeptides bind to asarcoma-associated polypeptide, or a unique fragment thereof.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, more preferably, an Fab or F(ab)₂ fragment of anantibody. Typically, the fragment includes a CDR3 region that isselective for the sarcoma-associated antigen. Any of the various typesof antibodies can be used for this purpose, including polyclonalantibodies, monoclonal antibodies, humanized antibodies, and chimericantibodies.

Thus, the invention provides agents which bind to sarcoma-associatedantigens encoded by sarcoma-associated nucleic acid molecules of theinvention, and in certain embodiments preferably to unique fragments ofthe sarcoma-associated polypeptides. Such binding partners can be usedin screening assays to detect the presence or absence of asarcoma-associated antigen and in purification protocols to isolate suchsarcoma-associated antigens. Likewise, such binding partners can be usedto selectively target drugs, toxins or other molecules (includingdetectable diagnostic molecules) to cells which expresssarcoma-associated antigens. In this manner, for example, cells presentin solid or non-solid tumors which express sarcoma-associated proteinscan be treated with cytotoxic compounds that are selective for thesarcoma-associated molecules (nucleic acids and/or antigens). Suchbinding agents also can be used to inhibit the native activity of thesarcoma-associated antigen, for example, to further characterize thefunctions of these molecules.

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering a protein, fragments of aprotein, cells expressing the protein or fragments thereof and the liketo an animal to induce polyclonal antibodies. The present invention alsoprovides methods of producing monoclonal antibodies to thesarcoma-associated molecules of the invention described herein. Theproduction of monoclonal antibodies is according to techniques wellknown in the art. As detailed herein, such antibodies may be used forexample to identify tissues expressing protein or to purify protein.Antibodies also may be coupled to specific labeling agents or imagingagents, including, but not limited to a molecule preferably selectedfrom the group consisting of fluorescent, enzyme, radioactive, metallic,biotin, chemiluminescent, bioluminescent, chromophore, or colored, etc.In some aspects of the invention, a label may be a combination of theforegoing molecule types.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)2 fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Fab fragments consist of acovalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd. The Fd fragments are the major determinant ofantibody specificity (a single Fd fragment may be associated with up toten different light chains without altering antibody specificity) and Fdfragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762, and 5,859,205.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. Following immunization of these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharn)), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (HAMA) responseswhen administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)2, Fab, Fv, and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)2 fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to sarcoma-associated antigens. These polypeptides maybe derived also from sources other than antibody technology. Forexample, such polypeptide binding agents can be provided by degeneratepeptide libraries which can be readily prepared in solution, inimmobilized form or as phage display libraries. Combinatorial librariesalso can be synthesized of peptides containing one or more amino acids.Libraries further can be synthesized of peptides and non-peptidesynthetic moieties.

The sarcoma-associated antigens of the invention can be used to screenpeptide libraries, including phage display libraries, to identify andselect peptide binding partners of the sarcoma-associated antigens ofthe invention. Such molecules can be used, as described, for screeningassays, for diagnostic assays, for purification protocols or fortargeting drugs, toxins and/or labeling agents (e.g., radioisotopes,fluorescent molecules, etc.) to cells which express sarcoma-associatedmolecules such as cancer cells which have aberrant sarcoma-associatedexpression.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe sarcoma-associated antigen. This process can be repeated throughseveral cycles of reselection of phage that bind to thesarcoma-associated polypeptide. Repeated rounds lead to enrichment ofphage bearing particular sequences. DNA sequence analysis can beconducted to identify the sequences of the expressed polypeptides. Theminimal linear portion of the sequence that binds to thesarcoma-associated polypeptide can be determined. One can repeat theprocedure using a biased library containing inserts containing part orall of the minimal linear portion plus one or more additional degenerateresidues upstream or downstream thereof. Yeast two-hybrid screeningmethods also may be used to identify polypeptides that bind to thesarcoma-associated antigens.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used to identify tissues with normal or aberrant expression of asarcoma-associated antigen. Antibodies also may be coupled to specificdiagnostic labeling agents for imaging of cells and tissues with normalor aberrant sarcoma-associated antigen expression or to therapeuticallyuseful agents according to standard coupling procedures. As used herein,“therapeutically useful agents” include any therapeutic molecule whichdesirably is targeted selectively to a cell or tissue selectively withan aberrant sarcoma-associated expression.

Diagnostic agents for in vivo use include, but are not limited to,barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium,diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoatesodium and radiodiagnostics including positron emitters such asfluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99, iodine-131 and indium-111, and nuclides for nuclearmagnetic resonance such as fluorine and gadolinium. Other diagnosticagents useful in the invention will be apparent to one of ordinary skillin the art.

The antibodies of the present invention can also be used totherapeutically target sarcoma-associated antigens. In a preferredembodiment, antibodies can be used to target antigens expressed on thecell surface, such as NY-SAR-35. These antibodies can be linked not onlyto a detectable marker but also an antitumor agent or animmunomodulator. Antitumor agents can include cytotoxic agents andagents that act on tumor neovasculature. Detectable markers include, forexample, radioactive or fluorescent markers. Cytotoxic agents includecytotoxic radionuclides, chemical toxins and protein toxins.

The cytotoxic radionuclide or radiotherapeutic isotope preferably is analpha-emitting isotope such as ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁴Raor ²²³Ra. Alternatively, the cytotoxic radionuclide may a beta-emittingisotope such as ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ⁶⁴Cu, ¹⁵³Sm or¹⁶⁶Ho. Further, the cytotoxic radionuclide may emit Auger and low energyelectrons and include the isotopes ¹²⁵I, ¹²³I or ⁷⁷Br.

Suitable chemical toxins or chemotherapeutic agents include members ofthe enediyne family of molecules, such as calicheamicin and esperamicin.Chemical toxins can also be taken from the group consisting ofmethotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine,mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.Other antineoplastic agents that may be conjugated to the anti-PSMAantibodies of the present invention include dolastatins (U.S. Pat. Nos.6,034,065 and 6,239,104) and derivatives thereof. Of particular interestis dolastatin 10(dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine) and thederivatives auristatin PHE(dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester)(Pettit, G. R. et al., Anticancer Drug Des. 13(4):243-277, 1998; Woyke,T. et al., Antimicrob. Agents Chemother. 45(12):3580-3584, 2001), andaurastatin B and the like. Toxins that are less preferred in thecompositions and methods of the invention include poisonous lectins,plant toxins such as ricin, abrin, modeccin, botulina and diphtheriatoxins. Of course, combinations of the various toxins could also becoupled to one antibody molecule thereby accommodating variablecytotoxicity. Other chemotherapeutic agents are known to those skilledin the art.

Agents that act on the tumor vasculature can include tubulin-bindingagents such as combrestatin A4 (Griggs et al., Lancet Oncol. 2:82,2001), angiostatin and endostatin (reviewed in Rosen, Oncologist 5:20,2000, incorporated by reference herein) and interferon inducible protein10 (U.S. Pat. No. 5,994,292). A number of antiangiogenic agentscurrently in clinical trials are also contemplated. Agents currently inclinical trials include: 2ME2, Angiostatin, Angiozyme, Anti-VEGF RhuMAb,Apra (CT-2584), Avicine, Benefin, BMS275291, Carboxyamidotriazole,CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC 412), CM101,Combretastatin A-4 Prodrug, EMD 121974, Endostatin, Flavopiridol,Genistein (GCP), Green Tea Extract, IM-862, ImmTher, Interferon alpha,Interleukin-12, Iressa (ZD1839), Marimastat, Metastat (Col-3),Neovastat, Octreotide, Paclitaxel, Penicillamine, Photofrin, Photopoint,PI-88, Prinomastat (AG-3340), PTK787 (ZK22584), RO317453, Solimastat,Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE 26644), Suramin(Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 and Vitaxin.additional antiangiogenic agents are described by Kerbel, J. Clin.Oncol. 19(18s):45s-51s, 2001, which is incorporated by reference herein.Immunomodulators suitable for conjugation to the antibodies includeα-interferon, γ-interferon, and tumor necrosis factor alpha (TNFα).

The coupling of one or more toxin molecules to the antibody isenvisioned to include many chemical mechanisms, for instance covalentbinding, affinity binding, intercalation, coordinate binding, andcomplexation. The toxic compounds used to prepare the immunotoxins areattached to the antibodies or antigen-binding fragments thereof bystandard protocols known in the art.

In other aspects of the invention, the sarcoma-associated molecules andthe antibodies and other binding molecules, as described herein, can beused for the treatment of disorders. When “disorder” is used herein, itrefers to any pathological condition where the sarcoma-associatedantigens are aberrantly expressed. An example of such a disorder iscancer, with sarcoma as a particular example. For human cancers,additional particular examples include synovial sarcoma, liposarcoma,neurosarcoma, chondrosarcoma, fibrosarcoma, Ewing sarcoma,leiomyosarcoma, osteosarcoma, rhabdomyosarcoma, malignant fibroushistocytoma, DFSP, leukemia, lymphoma, gastric cancer, glioma, bladdercancer, breast cancer, ovarian cancer, renal cancer, lung cancer, coloncancer, prostate cancer, esophageal cancer, melanoma and hepatoma.

Conventional treatment for cancer may include, but is not limited to:surgical intervention, chemotherapy, radiotherapy, and adjuvant systemictherapies. In one aspect of the invention, treatment may includeadministering binding polypeptides such as antibodies that specificallybind to the sarcoma-associated antigen. These binding polypeptides canbe optionally linked to one or more detectable markers, antitumor agentsor immunomodulators as described above.

Cancer treatment, in another aspect of the invention may includeadministering an antisense molecules or RNAi molecules to reduceexpression level and/or function level of sarcoma-associatedpolypeptides of the invention in the subject in cancers where asarcoma-associated molecule is up-regulated. The use of RNA interferenceor “RNAi” involves the use of double-stranded RNA (dsRNA) to block geneexpression. (see: Sui, G, et al, Proc Natl. Acad. Sci. U.S.A.99:5515-5520, 2002). Methods of applying RNAi strategies in embodimentsof the invention would be understood by one of ordinary skill in theart.

Sarcoma-associated polypeptides as described herein, can also be used inone aspect of the invention to induce or enhance an immune response.Some therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of antigenpresenting cells, such as cancer cells which present one or moresarcoma-associated antigens of the invention. One such approach is theadministration of autologous CTLs specific to a sarcoma-associatedantigen/MHC complex to a subject with abnormal cells of the phenotype atissue. It is within the ability of one of ordinary skill in the art todevelop such CTLs in vitro. An example of a method for T celldifferentiation is presented in International Application numberPCT/US96/05607. Generally, a sample of cells taken from a subject, suchas blood cells, are contacted with a cell presenting the complex andcapable of provoking CTLs to proliferate. The target cell can be atransfectant, such as a COS cell. These transfectants present thedesired complex of their surface and, when combined with a CTL ofinterest, stimulate its proliferation. COS cells are widely available,as are other suitable host cells. Specific production of CTL clones iswell known in the art. The clonally expanded autologous CTLs then areadministered to the subject.

Another method for selecting antigen-specific CTL clones has recentlybeen described (Altman et al., Science 274:94-96, 1996; Dunbar et al.,Curr. Biol. 8:413-416, 1998), in which fluorogenic tetramers of MHCclass I molecule/peptide complexes are used to detect specific CTLclones. Briefly, soluble MHC class I molecules are folded in vitro inthe presence of β₂-microglobulin and a peptide antigen which binds theclass I molecule. After purification, the MHC/peptide complex ispurified and labeled with biotin. Tetramers are formed by mixing thebiotinylated peptide-MHC complex with labeled avidin (e.g.phycoerythrin) at a molar ratio or 4:1. Tetramers are then contactedwith a source of CTLs such as peripheral blood or lymph node. Thetetramers bind CTLs which recognize the peptide antigen/MHC class Icomplex. Cells bound by the tetramers can be sorted by fluorescenceactivated cell sorting to isolate the reactive CTLs. The isolated CTLsthen can be expanded in vitro for use as described herein.

To detail a therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al, Eur. J. Immunol. 21: 1403-1410, 1991; Kast etal., Cell 59: 603-614, 1989), cells presenting the desired complex(e.g., dendritic cells) are combined with CTLs leading to proliferationof the CTLs specific thereto. The proliferated CTLs are thenadministered to a subject with a cellular abnormality which ischaracterized by certain of the abnormal cells presenting the particularcomplex. The CTLs then lyse the abnormal cells, thereby achieving thedesired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/cancer associated antigencomplex. This can be determined very easily, as the art is very familiarwith methods for identifying cells which present a particular HLAmolecule, as well as how to identify cells expressing DNA of thepertinent sequences, in this case a sarcoma-associated antigen sequence.Once cells presenting the relevant complex are identified via theforegoing screening methodology, they can be combined with a sample froma patient, where the sample contains CTLs. If the complex presentingcells are lysed by the mixed CTL sample, then it can be assumed that asarcoma-associated antigen is being presented, and the subject is anappropriate candidate for the therapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach is the use of non-proliferativecells expressing the complex. The cells used in this approach may bethose that normally express the complex, such as irradiated tumor cellsor cells transfected with one or both of the genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting MHC molecule). Chen et al. (Proc. Natl. Acad. Sci. USA 88:110-114, 1991) exemplifies this approach, showing the use of transfectedcells expressing HPV E7 peptides in a therapeutic regime. Various celltypes may be used. Similarly, vectors carrying one or both of the genesof interest may be used. Viral or bacterial vectors are especiallypreferred. For example, nucleic acids which encode a sarcoma-associatedpolypeptide may be operably linked to promoter and enhancer sequenceswhich direct expression of the sarcoma-associated antigen polypeptide incertain tissues or cell types. The nucleic acid may be incorporated intoan expression vector.

Expression vectors may be unmodified extrachromosomal nucleic acids,plasmids or viral genomes constructed or modified to enable insertion ofexogenous nucleic acids, such as those encoding sarcoma-associatedantigen, as described elsewhere herein. Nucleic acids encoding asarcoma-associated antigen also may be inserted into a retroviralgenome, thereby facilitating integration of the nucleic acid into thegenome of the target tissue or cell type. In these systems, the gene ofinterest is carried by a microorganism, e.g., a Vaccinia virus, poxvirus, herpes simplex virus, retrovirus or adenovirus, and the materialsde facto “infect” host cells. The cells which result present the complexof interest, and are recognized by autologous CTLs, which thenproliferate.

A similar effect can be achieved by combining the sarcoma-associatedpolypeptide or a stimulatory fragment thereof with an adjuvant tofacilitate incorporation into antigen presenting cells in vivo. Thesarcoma-associated polypeptide is processed to yield the peptide partnerof the MHC molecule while a sarcoma-associated fragment may be presentedwithout the need for further processing. Generally, subjects can receivean intradermal injection of an effective amount of thesarcoma-associated antigen. Initial doses can be followed by boosterdoses, following immunization protocols standard in the art. Preferredsarcoma-associated antigens include those found to react with allogeneiccancer antisera, shown in the examples below.

The invention involves the use of various materials disclosed herein to“immunize” subjects or as “vaccines”. As used herein, “immunization” or“vaccination” means increasing or activating an immune response againstan antigen. It does not require elimination or eradication of acondition but rather contemplates the clinically favorable enhancementof an immune response toward an antigen. Generally accepted animalmodels, can be used for testing of immunization against cancer using asarcoma-associated nucleic acid. For example, human cancer cells can beintroduced into a mouse to create a tumor, and one or moresarcoma-associated nucleic acids can be delivered by the methodsdescribed herein. The effect on the cancer cells (e.g., reduction oftumor size) can be assessed as a measure of the effectiveness of thesarcoma-associated nucleic acid immunization. Of course, testing of theforegoing animal model using more conventional methods for immunizationinclude the administration of one or more sarcoma-associatedpolypeptides or fragments derived therefrom, optionally combined withone or more adjuvants and/or cytokines to boost the immune response.

Methods for immunization, including formulation of a vaccine compositionand selection of doses, route of administration and the schedule ofadministration (e.g. primary and one or more booster doses), are wellknown in the art. The tests also can be performed in humans, where theend point is to test for the presence of enhanced levels of circulatingCTLs against cells bearing the antigen, to test for levels ofcirculating antibodies against the antigen, to test for the presence ofcells expressing the antigen and so forth.

As part of the immunization compositions, one or more sarcoma-associatedpolypeptides or immunogenic fragments thereof are administered with oneor more adjuvants to induce an immune response or to increase an immuneresponse. An adjuvant is a substance incorporated into or administeredwith antigen which potentiates the immune response. Adjuvants mayenhance the immunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., Mol. Cells. 7:178-186, 1997); incomplete Freund'sadjuvant; complete Freund's adjuvant; montanide; alum; CpGoligonucleotides (see e.g. Kreig et al., Nature 374:546-9, 1995); andvarious water-in-oil emulsions prepared from biodegradable oils such assqualene and/or tocopherol. Preferably, the antigens are administeredmixed with a combination of DQS21/MPL. The ratio of DQS21 to MPLtypically will be about 1:10 to 10:1, preferably about 1:5 to 5:1 andmore preferably about 1:1. Typically for human administration, DQS21 andMPL will be present in a vaccine formulation in the range of about 1 μgto about 100 μg. Other adjuvants are known in the art and can be used inthe invention (see, e.g. Goding, Monoclonal Antibodies: Principles andPractice, 2nd Ed., 1986). Methods for the preparation of mixtures oremulsions of polypeptide and adjuvant are well known to those of skillin the art of vaccination.

Other agents which stimulate the immune response of the subject can alsobe administered to the subject. For example, other cytokines are alsouseful in vaccination protocols as a result of their lymphocyteregulatory properties. Many other cytokines useful for such purposeswill be known to one of ordinary skill in the art, includinginterleukin-12 (IL-12) which has been shown to enhance the protectiveeffects of vaccines (see, e.g., Science 268:1432-1434, 1995), GM-CSF andIL-18. Thus cytokines can be administered in conjunction with antigensand adjuvants to increase the immune response to the antigens.

There are a number of immune response potentiating compounds that can beused in vaccination protocols. These include costimulatory moleculesprovided in either protein or nucleic acid form. Such costimulatorymolecules include the B7-1 and B7-2 (CD80 and CD86 respectively)molecules which are expressed on dendritic cells (DC) and interact withthe CD28 molecule expressed on the T cell. This interaction providescostimulation (signal 2) to an antigen/MHC/TCR stimulated (signal 1) Tcell, increasing T cell proliferation and effector function. 17 alsointeracts with CTLA4 (CD152) on T cells and studies involving CTLA4 andB7 ligands indicate that the B7-CTLA4 interaction can enhance antitumorimmunity and CTL proliferation (Zheng P., et al. Proc. Natl. Acad. Sci.USA 95 (11):6284-6289 (1998)).

B7 typically is not expressed on tumor cells so they are not efficientantigen presenting cells (APCs) for T cells. Induction of B7 expressionwould enable the tumor cells to stimulate more efficiently CTLproliferation and effector function. A combination of B7/IL-6/IL-12costimulation has been shown to induce IFN-gamma and a Th1 cytokineprofile in the T cell population leading to further enhanced T cellactivity (Gajewski et al., J. Immunol, 154:5637-5648 (1995)). Tumor celltransfection with B7 has been discussed in relation to in vitro CTLexpansion for adoptive transfer immunotherapy by Wang et al., (J.Immunol., 19:1-8 (1986)). Other delivery mechanisms for the B7 moleculewould include nucleic acid (naked DNA) immunization (Kim J., et al. Nat.Biotechnol., 15:7:641-646 (1997)) and recombinant viruses such as adenoand pox (Wendtner et al., Gene Ther., 4:7:726-735 (1997)). These systemsare all amenable to the construction and use of expression cassettes forthe coexpression of B7 with other molecules of choice such as theantigens or fragment(s) of antigens discussed herein (includingpolytopes) or cytokines. These delivery systems can be used forinduction of the appropriate molecules in vitro and for in vivovaccination situations. The use of anti-CD28 antibodies to directlystimulate T cells in vitro and in vivo could also be considered.Similarly, the inducible co-stimulatory molecule ICOS which induces Tcell responses to foreign antigen could be modulated, for example, byuse of anti-ICOS antibodies (Hutloff et al., Nature 397:263-266, 1999).

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCsand some tumor cells and interacts with CD2 expressed on T cells. Thisinteraction induces T cell IL-2 and IFN-gamma production and can thuscomplement but not substitute, the B7/CD28 costimulatory interaction(Parra et al., J. Immunol., 158:637-642 (1997), Fenton et al., J.Immunother., 21:2:95-108 (1998)).

Lymphocyte function associated antigen-1 (LFA-1) is expressed onleukocytes and interacts with ICAM-1 expressed on APCs and some tumorcells. This interaction induces T cell IL-2 and IFN-gamma production andcan thus complement but not substitute, the B7/CD28 costimulatoryinteraction (Fenton et al., J. Immunother., 21:2:95-108 (1998)). LFA-1is thus a further example of a costimulatory molecule that could beprovided in a vaccination protocol in the various ways discussed abovefor B7.

Complete CTL activation and effector function requires Th cell helpthrough the interaction between the Th cell CD40L (CD40 ligand) moleculeand the CD40 molecule expressed by DCs (Ridge et al., Nature, 393:474(1998), Bennett et al., Nature, 393:478 (1998), Schoenberger et al.,Nature, 393:480 (1998)). This mechanism of this costimulatory signal islikely to involve upregulation of B7 and associated IL-6/IL-12production by the DC (APC). The CD40-CD40L interaction thus complementsthe signal 1 (antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.

The use of anti-CD40 antibodies to stimulate DC cells directly, would beexpected to enhance a response to tumor antigens which are normallyencountered outside of an inflammatory context or are presented bynon-professional APCs (tumor cells). In these situations Th help and B7costimulation signals are not provided.

The invention contemplates delivery of nucleic acids, polypeptides orfragments thereof for vaccination. Delivery of polypeptides andfragments thereof can be accomplished according to standard vaccinationprotocols which are well known in the art. In another embodiment, thedelivery of nucleic acid is accomplished by ex vivo methods, i.e. byremoving a cell from a subject, genetically engineering the cell toinclude a sarcoma-associated polypeptide, and reintroducing theengineered cell into the subject. One example of such a procedure isoutlined in U.S. Pat. No. 5,399,346 and in exhibits submitted in thefile history of that patent, all of which are publicly availabledocuments. In general, it involves introduction in vitro of a functionalcopy of a gene into a cell(s) of a subject, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivonucleic acid delivery using vectors such as viruses and targetedliposomes also is contemplated according to the invention.

A virus vector for delivering a nucleic acid encoding asarcoma-associated polypeptide is selected from the group consisting ofadenoviruses, adeno-associated viruses, poxviruses including vacciniaviruses and attenuated poxviruses, Semliki Forest virus, Venezuelanequine encephalitis virus, retroviruses, Sindbis virus, and Tyvirus-like particle. Examples of viruses and virus-like particles whichhave been used to deliver exogenous nucleic acids include:replication-defective adenoviruses (e.g., Xiang et al., Virology219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997;Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus(Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicatingretrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replicationdefective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA92:3009-3013, 1995), canarypox virus and highly attenuated vacciniavirus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353,1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.Stand. 82:55-63, 1994), Venezuelan equine encephalitis virus (Davis etal., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al.,Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp et al.,Eur. J. Immunol. 26:1951-1959, 1996). A preferred virus vector is anadenovirus.

Preferably the foregoing nucleic acid delivery vectors: (1) containexogenous genetic material that can be transcribed and translated in amammalian cell and that can induce an immune response in a host, and (2)contain on a surface a ligand that selectively binds to a receptor onthe surface of a target cell, such as a mammalian cell, and therebygains entry to the target cell.

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection or infection with the foregoingviruses including the nucleic acid of interest, liposome mediatedtransfection, and the like. For certain uses, it is preferred to targetthe nucleic acid to particular cells. In such instances, a vehicle usedfor delivering a nucleic acid of the invention into a cell (e.g., aretrovirus, or other virus; a liposome) can have a targeting moleculeattached thereto. For example, a molecule such as an antibody specificfor a surface membrane protein on the target cell or a ligand for areceptor on the target cell can be bound to or incorporated within thenucleic acid delivery vehicle. Preferred antibodies include antibodieswhich selectively bind a sarcoma-associated antigen, alone or as acomplex with a MHC molecule. Especially preferred are monoclonalantibodies. Where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane proteinassociated with endocytosis may be incorporated into the liposomeformulation for targeting and/or to facilitate uptake. Such proteinsinclude capsid proteins or fragments thereof tropic for a particularcell type, antibodies for proteins which undergo internalization incycling, proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfully to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

According to a further aspect of the invention, compositions containingthe nucleic acid molecules, proteins, and binding polypeptides of theinvention are provided. The compositions contain any of the foregoingtherapeutic agents in an optional pharmaceutically acceptable carrier.Thus, in a related aspect, the invention provides a method for forming amedicament that involves placing a therapeutically effective amount ofthe therapeutic agent in the pharmaceutically acceptable carrier to formone or more doses. The effectiveness of treatment or prevention methodsof the invention can be determined using standard diagnostic methodsdescribed herein.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The term “physiologicallyacceptable” refers to a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism. Thecharacteristics of the carrier will depend on the route ofadministration. Physiologically and pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials which are well known in the art. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intratumoral, intraperitoneal, intramuscular, intracavity, subcutaneous,or transdermal. When antibodies are used therapeutically, a preferredroute of administration is by pulmonary aerosol. Techniques forpreparing aerosol delivery systems containing antibodies are well knownto those of skill in the art. Generally, such systems should utilizecomponents which will not significantly impair the biological propertiesof the antibodies, such as the paratope binding capacity (see, forexample, Sciarra and Cutie, “Aerosols,” in Remington's PharmaceuticalSciences, 18th edition, 1990, pp 1694-1712). Those of skill in the artcan readily determine the various parameters and conditions forproducing antibody aerosols without undue experimentation. When usingantisense preparations of the invention, slow intravenous administrationis preferred.

The compositions of the invention are administered in effective amounts.An “effective amount” is that amount of a sarcoma-associated polypeptidecomposition that alone, or together with further doses, produces thedesired response, e.g. increases an immune response to thesarcoma-associated polypeptide. In the case of treating a particulardisease or condition characterized by expression of one or moresarcoma-associated polypeptides, such as cancer, the desired response isinhibiting the progression of the disease. This may involve only slowingthe progression of the disease temporarily, although more preferably, itinvolves halting the progression of the disease permanently. This can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein. The desired response totreatment of the disease or condition also can be delaying the onset oreven preventing the onset of the disease or condition.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the individual components or combinations thereofbe used, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of sarcoma-associatedpolypeptide or nucleic acid encoding sarcoma-associated polypeptide forproducing the desired response in a unit of weight or volume suitablefor administration to a patient. The response can, for example, bemeasured by determining the immune response following administration ofthe sarcoma-associated polypeptide composition via a reporter system bymeasuring downstream effects such as gene expression, or by measuringthe physiological effects of the sarcoma-associated polypeptidecomposition, such as regression of a tumor or decrease of diseasesymptoms. Other assays will be known to one of ordinary skill in the artand can be employed for measuring the level of the response.

The doses of sarcoma-associated polypeptide compositions (e.g.,polypeptide, peptide, antibody, cell or nucleic acid) administered to asubject can be chosen in accordance with different parameters, inparticular in accordance with the mode of administration used and thestate of the subject. Other factors include the desired period oftreatment. In the event that a response in a subject is insufficient atthe initial doses applied, higher doses (or effectively higher doses bya different, more localized delivery route) may be employed to theextent that patient tolerance permits.

In general, for treatments for eliciting or increasing an immuneresponse, doses of sarcoma-associated antigen are formulated andadministered in doses between 1 ng and 1 mg, and preferably between 10ng and 100 μg, according to any standard procedure in the art. Wherenucleic acids encoding sarcoma-associated polypeptides or variantsthereof are employed, doses of between 1 ng and 0.1 mg generally will beformulated and administered according to standard procedures. Otherprotocols for the administration of sarcoma-associated polypeptidecompositions will be known to one of ordinary skill in the art, in whichthe dose amount, schedule of injections, sites of injections, mode ofadministration (e.g., intra-tumoral) and the like vary from theforegoing. Administration of sarcoma-associated polypeptide compositionsto mammals other than humans, e.g. for testing purposes or veterinarytherapeutic purposes, is carried out under substantially the sameconditions as described above.

Where sarcoma-associated polypeptides are used for vaccination, modes ofadministration which effectively deliver the sarcoma-associatedpolypeptide and adjuvant, such that an immune response to thepolypeptide is increased, can be used. For administration of asarcoma-associated polypeptide in adjuvant, preferred methods includeintradermal, intravenous, intramuscular and subcutaneous administration.Although these are preferred embodiments, the invention is not limitedby the particular modes of administration disclosed herein. Standardreferences in the art (e.g., Remington's Pharmaceutical Sciences, 18thedition, 1990) provide modes of administration and formulations fordelivery of immunogens with adjuvant or in a non-adjuvant carrier.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, and lactated Ringer's or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases, and the like.

The pharmaceutical agents of the invention may be administered alone, incombination with each other, and/or in combination with otheranti-cancer drug therapies and/or treatments. These therapies and/ortreatments may include, but are not limited to: surgical intervention,chemotherapy, radiotherapy, and adjuvant systemic therapies.

The invention also provides a pharmaceutical kit comprising one or morecontainers comprising one or more of the pharmaceutical compounds oragents of the invention. Additional materials may be included in any orall kits of the invention, and such materials may include, but are notlimited to buffers, water, enzymes, tubes, control molecules, etc. Thekit may also include instructions for the use of the one or morepharmaceutical compounds or agents of the invention for the treatment ofcancer.

The invention includes kits for assaying the presence ofsarcoma-associated antigens and/or antibodies that specifically bind tosarcoma-associated polypeptides. An example of such a kit may includethe above-mentioned polypeptides bound to a substrate, for example adipstick, which is dipped into a blood or body fluid sample of asubject. The surface of the substrate may then be processed usingprocedures well known to those of skill in the art, to assess whetherspecific binding occurred between the polypeptides and agents (e.g.antibodies) in the subject's sample. For example, procedures mayinclude, but are not limited to, contact with a secondary antibody, orother method that indicates the presence of specific binding.

Another example of a kit may include an antibody or antigen-bindingfragment thereof, that binds specifically to a sarcoma-associatedantigen. The antibody or antigen-binding fragment thereof, may beapplied to a tissue or cell sample from a patient with cancer and thesample then processed to assess whether specific binding occurs betweenthe antibody and an antigen or other component of the sample. Inaddition, the antibody or antigen-binding fragment thereof, may beapplied to a body fluid sample, such as serum, from a subject, eithersuspected of having cancer, diagnosed with cancer, or believed to befree of cancer. As will be understood by one of skill in the art, suchbinding assays may also be performed with a sample or object contactedwith an antibody and/or sarcoma-associated antigen that is in solution,for example in a 96-well plate or applied directly to an object surface.

Another example of a kit of the invention is a kit that providescomponents necessary to determine the level of expression of one or moresarcoma-associated nucleic acid molecules of the invention. Suchcomponents may include primers useful for amplification of one or moresarcoma-associated nucleic acid molecules and/or other chemicals for PCRamplification.

Another example of a kit of the invention is a kit that providescomponents necessary to determine the level of expression of one or moresarcoma-associated nucleic acid molecules of the invention using amethod of hybridization.

The foregoing kits can include instructions or other printed material onhow to use the various components of the kits for diagnostic purposes.

The invention further includes nucleic acid or protein microarrays(including antibody arrays) for the analysis of expression ofsarcoma-associated antigens or nucleic acids encoding such antigens. Inthis aspect of the invention, standard techniques of microarraytechnology are utilized to assess expression of the sarcoma-associatedantigens and/or identify biological constituents that bind suchantigens. The constituents of biological samples include antibodies,lymphocytes (particularly T lymphocytes), and the like. Microarraysubstrates include but are not limited to glass, silica,aluminosilicates, borosilicates, metal oxides such as alumina and nickeloxide, various clays, nitrocellulose, or nylon. The microarraysubstrates may be coated with a compound to enhance synthesis of a probe(peptide or nucleic acid) on the substrate. Coupling agents or groups onthe substrate can be used to covalently link the first nucleotide oramino acid to the substrate. A variety of coupling agents or groups areknown to those of skill in the art. Peptide or nucleic acid probes thuscan be synthesized directly on the substrate in a predetermined grid.Alternatively, peptide or nucleic acid probes can be spotted on thesubstrate, and in such cases the substrate may be coated with a compoundto enhance binding of the probe to the substrate. In these embodiments,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, preferably utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate. Nucleic acid probes preferably are linked using UVirradiation or heat.

Protein microarray technology, which is also known by other namesincluding protein chip technology and solid-phase protein arraytechnology, is well known to those of ordinary skill in the art and isbased on, but not limited to, obtaining an array of identified peptidesor proteins on a fixed substrate, binding target molecules or biologicalconstituents to the peptides, and evaluating such binding. See, e.g., G.MacBeath and S. L. Schreiber, “Printing Proteins as Microarrays forHigh-Throughput Function Determination,” Science 289(5485):1760-1763,2000.

Targets are peptides or proteins and may be natural or synthetic. Thetissue may be obtained from a subject or may be grown in culture (e.g.from a cell line).

In some embodiments of the invention, one or more control peptide orprotein molecules are attached to the substrate. Preferably, controlpeptide or protein molecules allow determination of factors such aspeptide or protein quality and binding characteristics, reagent qualityand effectiveness, hybridization success, and analysis thresholds andsuccess.

Nucleic acid arrays, particularly arrays that bind sarcoma-associatedantigens, also can be used for diagnostic applications, such as foridentifying subjects that have a condition characterized by aberrantsarcoma-associated antigen expression. Nucleic acid microarraytechnology, which is also known by other names including: DNA chiptechnology, gene chip technology, and solid-phase nucleic acid arraytechnology, is well known to those of ordinary skill in the art and isbased on, but not limited to, obtaining an array of identified nucleicacid probes on a fixed substrate, labeling target molecules withreporter molecules (e.g., radioactive, chemiluminescent, or fluorescenttags such as fluorescein, Cye3-dUTP, or Cye5-dUTP), hybridizing targetnucleic acids to the probes, and evaluating target-probe hybridization.A probe with a nucleic acid sequence that perfectly matches the targetsequence will, in general, result in detection of a strongerreporter-molecule signal than will probes with less perfect matches.Many components and techniques utilized in nucleic acid microarraytechnology are presented in The Chipping Forecast, Nature Genetics, Vol.21, January 1999, the entire contents of which is incorporated byreference herein.

According to the invention, probes are selected from the group ofnucleic acids including, but not limited to: DNA, genomic DNA, cDNA, andoligonucleotides; and may be natural or synthetic. Oligonucleotideprobes preferably are 20 to 25-mer oligonucleotides and DNA/cDNA probespreferably are 500 to 5000 bases in length, although other lengths maybe used. Appropriate probe length may be determined by one of ordinaryskill in the art by following art-known procedures. In one embodiment,preferred probes are sets of one or more of the sarcoma-associatednucleic acid molecules as described herein. Probes may be purified toremove contaminants using standard methods known to those of ordinaryskill in the art such as gel filtration or precipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or oligonucleotide to thesubstrate. These agents or groups may include, for example, amino,hydroxy, bromo, and carboxy groups. These reactive groups are preferablyattached to the substrate through a hydrocarbyl radical such as analkylene or phenylene divalent radical, one valence position occupied bythe chain bonding and the remaining attached to the reactive groups.These hydrocarbyl groups may contain up to about ten carbon atoms,preferably up to about six carbon atoms. Alkylene radicals are usuallypreferred containing two to four carbon atoms in the principal chain.These and additional details of the process are disclosed, for example,in U.S. Pat. No. 4,458,066, which is incorporated by reference in itsentirety.

In one embodiment, nucleic acid probes are synthesized directly on thesubstrate in a predetermined grid pattern using methods such aslight-directed chemical synthesis, photochemical deprotection, ordelivery of nucleotide precursors to the substrate and subsequent probeproduction.

Targets for microarrays are nucleic acids selected from the group,including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and maybe natural or synthetic. In all embodiments, nucleic acid targetmolecules from human tissue are preferred. The tissue may be obtainedfrom a subject or may be grown in culture (e.g. from a cell line).

In embodiments of the invention one or more control nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as nucleic acidquality and binding characteristics, reagent quality and effectiveness,hybridization success, and analysis thresholds and success. Controlnucleic acids may include but are not limited to expression products ofgenes such as housekeeping genes or fragments thereof.

EXAMPLES Materials and Methods

Cell Lines, Tissues, Sera and RNA

SW1045, SW982, and Fuji synovial sarcoma cell lines were obtained fromthe cell repository of the Ludwig Institute for Cancer Research (LICR),New York Branch at the Memorial Sloan-Kettering Cancer Center. Tumortissues and sera were obtained from Memorial Sloan-Kettering CancerCenter, Weill Medical College of Cornell University and Aichi CancerCenter Research Center, Nagoya Japan. Normal tissue RNA preparationswere purchased from Clontech laboratories Incorporated (Palo Alto,Calif.) and Ambion Incorporated (Austin, Tex.). Total RNA from tumortissues was prepared by the guanidinium thiocyanate method.

SEREX Analysis of cDNA Expression Libraries

Poly(A)+ RNA from two sarcoma cell lines, SW1045 and SW982, was preparedusing the Fast Track mRNA Purification Kit (Invitrogen, LifeTechnologies, Carlsbad, Calif.). Poly(A)+ RNA from normal testis waspurchased from CLONTECH. Separate cDNA libraries were constructed foreach of these in the ZAP Express vector (Stratagene, La Jolla, Calif.)according to the manufacturer's instructions using 5 μg polyA+ mRNA.Libraries containing 1−2×10⁶ primary recombinants were obtained and werenot amplified before immunoscreening.

To remove serum antibodies reactive with vector-related antigens, serawas absorbed against E. coli/bacteriophage lysates prepared in thefollowing manner. Wild-type lambda ZAP Express bacteriophage at aconcentration of 5,000 pfu (plaque-forming units) per 15 cm plate wereamplified in E. coli XL1 Blue MRF′ overnight in 100 ml NZY/0.7% agarose.10 ml of binding buffer (0.1M NaHCO3, pH 8.3) was then added to theplates, and the plates were gently agitated at 4° C. for 15 hours. Theresultant supernatants were collected and residual E. coli were lysed bysonication. The lysates were then coupled to CNBr-Sepharose 4B (AmershamPharmacia Biotech, Piscataway, N.J.) according to the manufacturer'sinstructions. Patient sera (1:10 dilution) were absorbed by batchabsorption with an equal volume of Sepharose 4B coupled E. coli/phagelysates at 4° C. for 6 hours. This procedure was repeated a total ofthree times and was followed by a 15 hour incubation with nitrocellulosefilters precoated with proteins derived from E. coli and E. coli/phagelysates. Library screenings were performed as previously described(Scanlan, M. J., et al. Characterization of human colon cancer antigensrecognized by autologous antibodies. Int. J. Cancer 1998; 76: 652-8.Scanlan, M. J., et al. Antigens recognized by autologous antibody inpatients with renal-cell carcinoma Int. J. Cancer 1999; 83: 456-64.) Atotal of five independent SEREX immunoscreenings of the cDNA librarieswere undertaken. Sera from 2 sarcoma patients were used independently,at a dilution of 1:200, to immunoscreen the cDNA libraries. A total of2.5−5.0×10⁵ or 1.75×10⁶ recombinants were screened per serum/cDNAlibrary combination. Serum reactive phage clones were converted toplasmid forms and subjected to DNA sequencing (Cornell University DNAServices, Ithaca, N.Y.).

Determination of Serum Antibody Reactivity

Two assays were used to determine serological reactivity, an ELISA-basedmethod and a bacteriophage expression method. With regard to CTantigens, serum antibody reactivity was determined by ELISA aspreviously described (Stockert E, et al. 1998. A survey of the humoralimmune response of cancer patients to a panel of human tumor antigens. JExp Med 187:1349-54.) Briefly, recombinant proteins (NY-ESO-1, SSX-2,MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7 and CT10) were produced in E.coli by transfection with pQE30 expression vectors (Qiagen, Chatsworth,Calif.) according to the manufacturer's protocol. 10 ng of recombinantprotein (1 μg/ml) was absorbed to TC microwell plates (Nalge NuncInternational Corp., Naperville, Ill.) for 15 hours at 4° C. Afterwashing with PBS, plates were then blocked with 2% BSA and incubatedwith diluted (1:100-1:25,000) patient sera for 2 hours at roomtemperature. Following a PBS wash step, 10 μl of a 1:5000 dilution ofalkaline phosphatase-conjugated goat anti-human IgG secondary antibody(Southern Biotechnology, Birmingham, Ala.) was added to each well andincubated for 1 hour at room temperature. Following a PBS wash step,plates were incubated with 10 ul/well Attophose substrate (JBLScientific, San Louis Obispo, Calif.) for 25 min, and the fluorescencewas then read by a Cyto-Fluor 2350 (Millipore, Bedford, Mass.).

In the case of SEREX-defined sarcoma antigens, a previously describedserum antibody detection array (SADA or spot immunoassay (Scanlan M J,et al. Humoral immunity to human breast cancer: antigen definition andquantitative analysis of mRNA expression. Cancer Immunity 1:4 [epub];Scanlan M J. et al. 2002. Cancer-Related Serological Recognition ofHuman Colon Cancer: Identification of Potential Diagnostic andImmunotherapeutic Targets. Cancer Res. 2002 Jul. 15; 16 (14): 4041-7.)was used to determine serological reactivity.

Preabsorbed serum samples from 39 sarcoma patients and 33 healthy blooddonors were evaluated for the presence of IgG antibody reactive to apanel of SEREX-defined sarcoma antigens, identified herein, in thefollowing manner. Precut nitrocellulose membranes (80×120 mm) wereprecoated with a layer (approximately 0.2 mm) of growth media (NZY/0.7%Agarose/2.5 mM isopropyl-β-D-thiogalactopyranoside) and placed on areservoir layer of NZY/0.7% Agarose in a 86×128 mm Omni Tray (NalgeNunc). 5.0×10³ pfu per μl of bacteriophage encoding individualSEREX-defined tumor antigens were mixed with an equal volume ofexponentially growing E. coli XL-1 Blue MRF′ and spotted (0.7 μlaliquots) on the precoated nitrocellulose membranes using a 96 pinreplicator (Nalge Nunc). Membranes were incubated for 15 hours at 37° C.and then processed as per the standard SEREX protocol (Scanlan, et al.,Int. J. Cancer 1998; 76: 652-8; Scanlan, et al., Int. J. Cancer 1999;83: 456-64). Briefly, plates were blocked in 0.5% non-fat dried milk;incubated in 10 ml of a 1:200 dilution of sera at room temperature for15 hours; and then incubated in a 1:3000 dilution of alkalinephosphatase conjugated, Fc fragment specific, goat anti-human IgG(Jackson Immunoresearch laboratories Inc., West Grove Pa.). Serum IgGreactivity was detected with the alkaline phosphatase substrate, 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate.

Reverse Transcriptase-PCR (RT-PCR) Analysis

The cDNA preparations used as templates in the RT-PCR reactions wereprepared using the Superscript first strand synthesis kit (InvitrogenLife Technologies, Carlsbad, Calif.) according to the manufacturer'sinstructions using 2.5 μg of total RNA. For evaluation of CT antigensexpression in sarcoma cell lines, PCR primers specific for NY-ESO-1,LAGE-1, MAGE-1, MAGE-3, MAGE-4, MAGE-10, SCP-1, BAGE, CT7, SSX-1, SSX-2,and SSX-4 were synthesized commercially (Invitrogen Life Technologies)using published primer sequences (van der Bruggen P, et al. 1991. A geneencoding an antigen recognized by cytolytic T lymphocytes on a humanmelanoma. Science 254:1643-47, Gaugler, B., et al. Human gene MAGE-3codes for an antigen recognized on a melanoma by autologous cytolytic Tlymphocytes. J. Exp. Med. 1994; 179: 921-30, Chen, Y.-T., et al. Atesticular antigen aberrantly expressed in human cancers detected byautologous antibody screening. Proc. Natl. Acad. Sci. USA. 1997; 94:1914-18; Boel, P., et al., and van der Bruggen, P. BAGE: a new geneencoding an antigen recognized on human melanomas by cytolytic Tlymphocytes. Immunity 1995; 2: 167-75. (PMID: 7895173); Sahin U, et al.1998. Expression of multiple cancer/testis antigens in breast cancer andmelanoma: basis for polyvalent CT vaccine strategies. Int J Cancer78:387-89, Lethe B, et al. 1998. LAGE-1, a new gene with tumorspecificity. Int. J. Cancer 76:903-8, Türeci Ö, et al. 1998. Expressionof SSX genes in human tumors. Int J Cancer 77:19-23, Gure A O, et al.1997. SSX: a multigene family with several members transcribed in normaltestis and human cancer. Int J Cancer 72:965-971). PCR primers specificfor SEREX-defined antigens were also synthesized commercially(Invitrogen Life Technologies) and their sequences are as follows:NY-SAR-12 forward, TggCgCAgAAAggAAAAggAAAAT (SEQ ID NO: 91); NY-SAR-12reverse, AgAggTAgCTggCAggATgTTAg (SEQ ID NO: 92); NY-SAR-35 forward,CTTggTgCgATCAgCCTTAT (SEQ ID NO: 93); NY-SAR-35 reverse,TTgATgCATgAAAACAgAACTC (SEQ ID NO: 94); NY-SAR-41 forward,AgAATTggCAgAggCTCgTCATCA (SEQ ID NO: 95); NY-SAR-41 reverse,TTCCAATTTTgCCTTCTCTAACTg (SEQ ID NO: 96); NY-SAR-73 forward,CCCggAgCACgTCgAggTCTAC (SEQ ID NO: 135); NY-SAR-73 reverse,ggTgAggggCCCAggAAgC (SEQ ID NO: 136); NY-SAR-78 forward,CACAATgTATCCTgTTgAAAg (SEQ ID NO: 137); NY-SAR-78 reverse,gAgATgATACATTCTTCCAg (SEQ ID NO: 138); NY-SAR-92 forward,CTTCCgCCAACTCCTCCTACC (SEQ ID NO: 139); NY-SAR-92 reverse,gATgCCCgTgTCTTgTCCTT (SEQ ID NO: 140); NY-SAR-96 forward,CACTAggCTgCTgAggAAgAT (SEQ ID NO: 141); NY-SAR-96 reverse,gTTTTggTgggCAgCATTgAg (SEQ ID NO: 142); NY-SAR-97 forward,ggACCACCCCAAATAgAA (SEQ ID NO: 143); NY-SAR-97 reverse,CCACCAgCTCAggAAgA (SEQ ID NO: 144); NY-SAR-110 forward,TCTgATggAgCggTgggATgC (SEQ ID NO: 145); NY-SAR-110 reverse,gTgTgCCTCggCTTCTTTCTTC (SEQ ID NO: 146).

RT-PCR was performed in the following manner. Twenty-five μl PCRreaction mixtures, consisting of 2 μl cDNA, 0.2 mM dNTP, 1.5 mM MgCl₂,0.25 μM gene specific forward and reverse primers, and 2.5 U PlatinumTaq DNA polymerase (Invitrogen Life Technologies), were heated to 94° C.for 2 min., followed by 35 thermal cycles of 94° C. for 30 seconds, 55°C. for 30 seconds and 72° C. for 1 min., and a final cycle of 94° C. for30 seconds, 55° C. for 30 seconds and 72° C. for 5 min. Thermal cyclingwas performed using a GeneAmp PCR System 9700 (Applied Biosystems,Foster City, Calif.). Resultant PCR products were analyzed in 2%Agarose/Tris-Acetate-EDTA gels.

Real-time Quantitative Reverse Transcription (RT)-PCR

The concentration of NY-SAR-35 mRNA transcripts in normal tissues wasmeasured by real-time RT-PCR using cDNA preparations derived from lungcancer specimens and 16 different normal tissues that had beennormalized for 6 housekeeping genes (Clontech). Gene-specific TaqManprobes and PCR primers were designed using Primer Express software (PEBiosystems, Foster City, Calif.). PCR reactions were prepared using 2.5μl of cDNA diluted in TaqMan PCR Master Mix (PE Biosystems) supplementedwith 200 nM 6-carboxy-fluorescein labeled gene-specific TaqMan probe,and a predetermined, optimum concentration of gene specific forward andreverse primers (300-900 nM). Triplicate PCR reactions were prepared foreach cDNA sample. PCR consisted of 40 cycles of 95° C. denaturation (15seconds) and 60° C. annealing/extension (60 seconds). Thermal cyclingand fluorescent monitoring were performed using an ABI 7700 sequenceanalyzer (PE Biosystems). The point at which the PCR product is firstdetected above a fixed threshold, termed cycle threshold (Ct), wasdetermined for each sample. The abundance of gene-specific transcriptsin normal tissues was determined by comparison with a standard curvegenerated from the Ct values of known concentrations of plasmid DNAtemplate encoding NY-SAR-35.

TaqMan primers were as follow: NY-SAR-35 forward, TggTgCgATCAgCCTTATCC(SEQ ID NO: 147); NY-SAR-35 reverse, CggTTCgCTCCTCCAgAA (SEQ ID) NO:148). TaqMan probe: NY-SAR-35, TgTCTgCCCATTTATTgCCgCTCTCT (SEQ ID NO:149).

Northern Blot Analysis.

A Northern blot containing poly A+ RNA (2 μg/lane) from various normaltissues was obtained commercially (Clontech). An NY-SAR-35 cDNA probe(bp 263-1029) was labeled using the Bright Star Psoralen-Biotin Kit(Ambion Inc., Austin, Tex.) and hybridized to the membrane for 15 hoursat 68° C. After washing, the hybridization signal was developed usingthe Bright Star Bio-Detect Kit, according to the manufacturer'sinstructions (Ambion).

Southern Blot Analysis

Genomic DNA was extracted from normal human testis, and samples (10 μg)were independently digested with EcoRI, HindIII, and BamHI at 37° C.overnight. The DNA was then separated on 0.7% agarose gel and blottedonto a nylon transfer membrane. An NY-SAR-35 cDNA probe (bp 252-1029)was radiolabeled with ³²P-dCTP using a random-primer DNA labeling kit(Roche Molecular Biochemicals, Indianapolis, Ind.). The blot washybridized to a ³²P labeled probe at 68° C. After 15 hours ofhybridization, the membrane was washed under high stringency conditions(0.1×SSC, 0.5% SDS at 60° C.) and exposed for autoradiography.

Example 1 Results Front the First Round of Immunoscreenings by SEREXAnalysis

Identification of Human Sarcoma Antigens by SEREX Analysis

Preliminary studies were carried out to determine optimum sources oftarget antigens and immunoreactive patient sera. Three sarcoma celllines were typed for expression of NY-ESO-1, LAGE-1, MAGE-1, MAGE-3,MAGE-4, MAGE-10, BAGE, SCP-1, CT7, SSX-1, SSX-2, and SSX-4 transcriptsby RT-PCR. As shown in Table 2, all 3 sarcoma cell lines expressed atleast one of the transcripts in this panel. Specifically, the SW982 andSW1045 synovial sarcoma cell lines expressed 8 and 10 of the 12 CTantigen transcripts in the panel, respectively, while Fuji synovialsarcoma cells expressed 4/12 CT antigen transcripts.

TABLE 2 Cancer/Testis antigen expression in sarcoma cell lines Cell LineCT Antigen SW982 (synovial) SW1045 (synovial) Fuji NY-ESO-1 + + + LAGE-1Neg + + MAGE-A1 + + Neg MAGE-A3 + + Neg MAGE-A4 + + MAGE-A10 + + NegBAGE + + Neg SCP-1 Neg Neg Neg CT7 + + Neg SSX1 Neg + Neg SSX2 Neg NegSSX4 + + Neg Totals 8/12 10/12 4/12

In order to identify a subset of sarcoma patients that are activelymounting an immune response against tumor antigens, sera from 54 sarcomapatients (various histologies) were tested by ELISA (Stockert E, et al.1998. A survey of the humoral immune response of cancer patients to apanel of human tumor antigens. J Exp Med 187:1349-54) for the presenceof antibodies against a panel of 8 CT antigens consisting of: NY-ESO-1,SSX-2, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7 and CT10. Only 2/54sarcoma patients, a malignant fibrous histocytoma (MFH) and fibrosarcomapatient (FS), had detectable serum antibodies against a CT antigen,while the remaining 52 patients lacked detectable anti-CT antigenantibodies. Both seropositive patients had antibodies to NY-ESO-1 butlacked antibodies to the other 7 CT antigens tested. Fibrosarcoma tissuefrom the NY-ESO-1 seropositive patient, FS, was available for CT antigentyping by RT-PCR and was found to express 11/12 different CT antigentranscripts (NY-ESO-1, LAGE-1, MAGE-A1, -A3, -A4, -A10, BAGE, CT7, SSX1,-2 and -4). Tissue from the NY-ESO-1 seropositive patient, MFH, was notavailable for CT antigen typing by RT-PCR.

Although it was determined that CT antigen expression is frequent insarcoma tissue, serum antibody responses were not as frequent. This lackof immunogenicity in sarcoma may be an indication of immune escape bysarcoma cells, whereby the immune system fails to recognize CT antigensand eliminate tumor cells expressing these antigens, resulting in theexpansion of a homogenous population CT antigen expressing sarcomacells. Relevant escape mechanisms include defective antigen presentation(Garrido F, Algarra I. MHC antigens and tumor escape from immunesurveillance. Adv Cancer Res 2001; 83:117-58) and/or production ofimmuno-inhibitory cytokines, such as TGF-β and IL-10 (Conrad C T, et al.Differential expression of transforming growth factor beta 1 andinterleukin 10 in progressing and regressing areas of primary melanoma.J Exp Clin Cancer Res 1999 Jun.; 18(2):225-32). It is also possible thathomogeneous NY-ESO-1 and MAGE expression in synovial sarcoma (JungbluthA A, et al. 2001. Monophasic and biphasic synovial sarcomasabundantly/express cancer/testis antigen NY-ESO-1 but not MAGE-A1 orCT7. Int J Cancer 94:252-6; Antonescu C R, et al. MAGE antigenexpression in monophasic and biphasic synovial sarcoma. Hum Pathol 2002February; 33(2):225-9), as opposed to heterogeneous CT antigenexpression observed in many other tumor types (Jungbluth A A, et al.2001. Immunohistochemical analysis of NY-ESO-1 antigen expression innormal and malignant human tissues. Int J Cancer 92:856-60; Jungbluth AA, et al. 2000. Expression of MAGE-antigens in normal tissues andcancer. Int J Cancer 85:460-5), may also be a contributing factor toimmune escape.

These 2 patients were chosen as the serum sources for SEREXimmunoscreening of cDNA libraries prepared from the SW982 and SW1045synovial sarcoma cell lines. A total of 4 SEREX immunoscreenings wereperformed, leading to the identification of 72 distinct sarcomaantigens, designated NY-SAR-1 through NY-SAR-72. As shown in Table 3,immunoscreening with sera from an NY-ESO-1 serum antibody positive MFHpatient led to the identification of 28 antigens, including 8overlapping antigens derived from both the SW982 and SW1045 cDNAlibraries, as well as 13 antigens derived solely from the SW982 cDNAlibrary, and 7 antigens derived solely from the SW1045 cDNA library.

Immunoscreening with sera from an NY-ESO-1 serum antibody positivefibrosarcoma patient defined 46 antigens, including 2 overlappingantigens derived from both the SW982 and SW1045 cDNA libraries, as wellas 25 antigens derived solely from the SW982 cDNA library, and 19antigens derived solely from the SW1045 cDNA library. There was littleoverlap between the antigens recognized by serum antibodies from the MFHand FS patients. Only three antigens, NY-SAR-1/TMF1, NY-SAR-4/FH andNY-SAR-17/LAGE-1 were identified with both the MFH and FS sera. Becauseserological reactivity to NY-ESO-1 was the criteria used in selectingsera for cDNA library screening, mutual immunoreactivity to the highlyhomologous (84% amino acid identity) NY-SAR-17/LAGE-1 antigen wasexpected, and, although not intending to be bound by a particulartheory, is likely to be due to shared epitopes. The 72 antigens (Tables4-6) represent 58 known proteins and 14 uncharacterized gene products.

TABLE 3 Immunoscreening of synovial sarcoma cDNA expression librarieswith allogeneic sarcoma patient sera Synovial Number of Total sarcomacDNA Number of different number of expression recombinants antigensdistinct Sarcoma Serum library screened identified antigens MalignantSW982   5 × 10⁵ 21 28 Fibrous SW1045   5 × 10⁵ 15 HistocytomaFibrosarcoma SW982 2.5 × 10⁵ 27 46 SW1045 2.5 × 10⁵ 21

TABLE 4 SEREX-defined sarcoma antigens: antigens reactive with sera frommultiple cancer patients Reactivity SEREX Database NY- Identity with IDNumber² of SAR- (Unigene Sarcoma Equivalent Isolate Antigen cluster)Sera Source of Reactive Sera¹ Tumor Source¹) 2 STAU 2/39 MFH (#3), OS(#2) 614 (PRC), 1273 (Hs.6113) (BC) 4 FH (Hs.75653) 5/39 MFH (#3), OS(#4, #7), No Match ES (#1), FS (#2) 12 NESG1 2/39 MFH (#3), LS (#4) NoMatch (Hs.158450) 13 ACTN1 1/39 MFH (#3) 855 (BC) (Hs.119000) 15 RBM61/39 MFH (#3) 76 (LC) (Hs.173993) 16 FLJ12785 1/39 MFH (#3) 756 (TALL)(Hs.192742) 17 LAGE-1a 2/39 MFH (#3), FS (#2) 1160 (BC) (Hs.87225) 18SSSCA1 1/39 MFH (#3) 1799 (CC) (Hs.25723) 28 MGC: 9727 1/39 MFH (#3) 71(BC) (Hs.11065) 30 SNK (Hs.3838) 2/39 FS (#2), RS (#1) No Match 44LGALS1 1/39 FS (#2) 704 (RC) (Hs.227751) 47 MIF (Hs.73798) 1/39 FS (#2)989 (MEL) 50 PYCR1 3/39 FS (#2), MFH (#2, #4) No Match (Hs.79217) 71None 1/39 FS (#2) 1938 (GL) (Hs.314941) 72 HSPE1 1/39 FS (#2) 882 (HC),1202 (Hs.1197) (MEL) Antigens did not react with sera from normal blooddonors (0/33). ¹Abbreviations: BC, breast cancer; CC, colon cancer; ES,Ewing sarcoma; FS, fibrosarcoma; GC, gastric cancer; GL, glioma; HC,hepatocellular carcinoma; LC, lung cancer; LS, leimyosarcoma; MEL,melanoma; MFH, malignant fibrous histocytoma; OC, ovarian cancer; OS,osteosarcoma; PRC, prostate cancer; RC, renal cancer; RS,rhabdomyosarcoma; TALL, T-cell acute lymphocytic leukemia. ²SEREXdatabase ID numbers from the LICR's SEREX database(licr.org/SEREX.html).

TABLE 5 SEREX-defined sarcoma antigens: antigens reactive with sera fromboth normal donors and sarcoma patients SEREX Database ID ReactivityReactivity NY- Number¹ of with with SAR- Identity Equivalent Isolate(Tumor Normal Sarcoma Antigen (Unigene cluster) Source²) Sera Sera 1TMF1 (Hs.267632) 246 (G), 1241 (BC) 2/33 3/39 3 KIAA1536 89 (BR) 2/333/39 (Hs.156667) 6 RHAMM 1513 (OC) 1/33 3/39 (Hs.72550) 7 PINCH 344(CC), 550 (GC), 1152 16/21  14/39  (Hs.112378) (RC), 1281 (BR) 10KIAA0603 No Match 11/33  4/39 (Hs.173802) 11 U2AF1RS2 430 (RC), 786(HD), 1236 6/33 17/39  (Hs.171909) (BC), 1334 (GC) 14 SC65 (Hs.207251)No Match 8/33 4/39 19 HEF1 (Hs.80261) 421 (RC) 3/33 7/39 22 NELIN NoMatch 4/33 19/39  (Hs.216381) 29 FLJ13441 974 (PC) 6/33 3/39 (Hs.232146)31 HUMAUANTIG 1017 (BC), 1331 (GC), 1475 2/33 6/39 (Hs.75528) (OC) 32PDAP1 No Match 4/33 8/39 (Hs.278426) 33 SURF6 No Match 2/33 2/39(Hs.274430) 41 None (Hs.166670) No Match 1/33 1/39 45 STIP1 (Hs.75612)430 (RC) 4/33 2/39 53 FXYD5 No Match 1/33 1/39 (Hs.333418) 54 LMOD1 NoMatch 7/33 13/39  (Hs.79386) 55 RBM10 No Match 1/33 1/39 (Hs.154583) 58LIP8 (Hs.348012) No Match 1/33 3/39 61 ZNF282 No Match 1/33 2/39(Hs.58167) 64 USP16 (Hs.99819) No Match 2/33 2/39 65 FDFT1 (Hs.48876) NoMatch 2/33 1/39 66 ROCK1 444 (RC) 1/33 1/39 (Hs.109450) 68 P38IP(Hs.333500) No Match 1/33 3/39 ¹The LICR's SEREX database ID numbersfrom licr.org/SEREX.html. ²Abbreviations: BC, breast cancer; CC, coloncancer; HD, Hodgkins disease; GC, gastric cancer; OC, ovarian cancer;PC, pancreatic cancer; RC, renal cancer.

TABLE 6 SEREX-defined sarcoma antigens: antigens reactive with sera froma single sarcoma patient NY- SAR- Gene Identity Antigen (UnigeneCluster) 5 TBC1D1(Hs.278586) 8 BIRC2 (Hs.289107) 9 ATP5B (Hs.25) 20TCEB3 (Hs.155202) 21 GTF3C3 (Hs.90847) 23 C20orf81 (Hs.29341) 24 None(not clustered) 25 PDE4DIP (Hs.265848) 26 PIASX-BETA (Hs.111323) 27FLJ10330(Hs.342307) 34 SEC23B (Hs.173497) 35 None (Hs.128580) 36SSX1(Hs.194759) 37 MP1 (Hs.260116) 38 HMG20B (Hs.32317) 39 PSMD4(Hs.148495) 40 INPP1 (Hs.32309) 42 BTG3 (Hs.77311) 43 SSX4 (Hs.278632)46 ARNTL2 (Hs.222024) 48 MGC20533 (Hs.69280) 49 EMK1 (Hs.157199) 51 EDF1(Hs.174050) 52 Actin (Hs.288061) 56 MLF1Hs.85195) 57 GCN5L2 (Hs.101067)59 UPF3B (Hs.103832) 60 EGLN1 (Hs.6523) 62 AD034(Hs.281397) 63USP19(Hs.301373) 67 LUC7L (Hs.16803) 69 ARL1 (Hs.242894) 70 RPL10A(Hs.334895) ¹Antigens reacted with sera from single sarcoma patient(1/39), but not with sera from normal individuals (0/33). The antigenslisted had no matches with existing entries in the SEREX database(licr.org/SEREX.html).

The nucleotide sequences of all uncharacterized gene products (NY-SAR-3,-10, -16,-22, -23, -24, -27, -28, -29, -35, -41, -48, -62, -71) havebeen deposited in the GenBank database (SEQ ID NOs: 1-14, respectively).The cDNA sequences encoding the 72 sarcoma antigens were also comparedto sequences deposited in the SEREX database accessible through awebsite of the Ludwig Institute for Cancer Research(licr.org/SEREX.html). Examination of this database revealed that 21 ofthe 72 sarcoma antigens defined in this study (29%) were also identifiedthrough SEREX analysis of other tumor types (Tables 4 and 5).

Reactivity Patterns of Sera from Normal Individuals and Cancer Patientswith SEREX-defined Sarcoma Antigens

To determine whether immune recognition of the isolated antigens wascancer-related, allogeneic sera samples obtained from 33 normal blooddonors and 39 sarcoma patients (various histologies) were tested forreactivity against the 72 sarcoma antigens defined in the current studyusing serum antibody detection arrays (SADA). Twenty-four of the 72antigens (33%) had a serological profile that was not restricted tocancer patients, as evidenced by their reactivity with normal sera.These antigens have been listed in Table 5.

Sera from two normal individuals and three sarcoma patients reacted withNY-SAR-1/TMF1, suggesting the reactivity was unrelated to cancers. Withone notable exception (NY-SAR-22/NELIN), the frequency of antibodyresponses to 23 of the 24 antigens associated with normal serareactivity was similar in normal blood donors and cancer patients. Inthe case of NY-SAR-22/NELIN (UniGene cluster Hs.216381), the frequencyof antibody responses was considerably higher in cancer patients, inwhich 19/39 (49%) of sarcoma patients and 4/33 (12%) of normalindividuals had a detectable antibody response. The remaining 48antigens had a cancer-related serological profile, reacting only withsera from cancer patients.

The 48 antigens having a cancer-related serological profile could besubdivided into 4 categories; a) antigens identified by serum from onlya single sarcoma patient; b) antigens that reacted with sera from asingle sarcoma patient and, as determined by an analysis of the SEREXdatabase, with sera from patients having other forms of cancer; c)antigens that reacted exclusively with sera from 2 or more sarcomapatients; and d) antigens that reacted with sera from 2 or more sarcomapatients and with sera from patients having other forms of cancer. Ofthe 48 antigens having a cancer-related serological profile, 33 antigensreacted with sera from a single sarcoma patient (Table 6).

As shown in Table 4, the remaining 15 antigens reacted with sera from 2or more cancer patients, but not with sera from normal individuals. Nineantigens reacted with sera from a single sarcoma patient, and with serafrom patients with other tumor types (NY-SAR-13, -15, -16, -18, -28,-44, -47, -71, -72). Four antigens reacted exclusively with sera from 2or more sarcoma patients (NY-SAR, 4, -12, -30, -50). The remaining twoantigens, NY-SAR-2/STAU and the CT antigen, NY-SAR-17/LAGE-1A, reactedwith sera from 2 or more sarcoma patients and with sera from patientswith other types of cancer. A cancer-related serological response toNY-SAR-4/FH occurred most frequently. In this case, serum samples from5/39 (13%) sarcoma patients were reactive with NY-SAR-4/FH, including2/10 sera samples from osteosarcoma patients, 1/6 sera samples frommalignant fibrous histiocytoma patients, 1/2 patients sera samples fromfibrosarcoma patients, and 1/7 sera samples from Ewing sarcoma patients.No serological responses to NY-SAR-4/FH were detected in normal blooddonors.

This serological response to NY-SAR-4/FH is of interest as germ-linemutations in the FH gene have been associated with a predisposition touterine and cutaneous leiomyomata and also renal cell carcinoma(Tomlinson I P, et al. Germline mutations in FH predispose to dominantlyinherited uterine fibroids, skin leiomyomata and papillary renal cellcancer. Nat Genet. 2002 Apr.; 30(4):406-10) and is a target of somaticmutation in sarcoma (Kiuru, M., et al. (2002) Cancer Res. 62, 455-44557)suggesting that the immune response is directed against mutatedepitopes.

Expression Patterns of mRNA Encoding Serologically Defined SarcomaAntigens in Normal and Malignant Tissues

A preliminary in silico mRNA expression profile of all gene productsidentified in this study was carried out based on the tissuedistribution of expressed sequence tags (ESTs) in the human ESTdatabase. Products with no EST matches, or those having EST matcheslimited to tumor tissue, fetal tissue, and/or less than 3 normal adulttissues were further examined by RT-PCR. Gene products with restrictedEST profiles include the three well-characterized cancer-testisantigens, LAGE-1/NY-SAR-17, NY-SAR-36/SSX1, and NY-SAR-43/SSX4, whichare expressed exclusively in normal testis and a range of differenttumor types (Lethe B, et al. 1998. LAGE-1, a new gene with tumorspecificity. Int. J. Cancer 76:903-8; Türeci Ö, et al. 1998. Expressionof SSX genes in human tumors. Int. J. Cancer 77:19-23; Gure A O, et al.1997. SSX: a multigene family with several members transcribed in normaltestis and human cancer. Int. J. Cancer 72:965-971), and 3 putativetissue restricted antigens, including a known gene product,nasopharyngeal specific protein 1 (NESG1)/NY-SAR-12 (Li Z, Yao K, Cao Y.Molecular cloning of a novel tissue-specific gene from humannasopharyngeal epithelium. Gene 1999 Sep. 3; 237(1):235-40), and 2uncharacterized gene products, NY-SAR-35 (UniGene cluster Hs.128580) andNY-SAR-41 (UniGene cluster Hs.166670). With the exception of serumreactivity to NY-SAR-41 occurring in 1/33 normal blood donors, thesedifferentially expressed antigens showed a cancer-related serologicalprofile.

As shown in FIG. 1A, mRNA expression patterns of NY-SAR-12, -35, and -41were examined in 17 different human tissues by RT-PCR, NESG1/NY-SAR-12mRNA was detected in normal placenta, testis, colon, lung, and ovary(0/12 other normal tissues). NY-SAR-35 mRNA was detected only in normaltestis (0/15 other normal tissues), while a lower molecular weighttranscript was detected in normal ovary. NY-SAR-41 was detected innormal testis, fetal brain, colon, lung, and bladder (0/12 other normaltissues). As shown in FIG. 1B, the testis restricted expression patternof NY-SAR-35 was confirmed by real time quantitative RT-PCR at 40amplification cycles. In these studies, NY-SAR-35 was expressed innormal testis at a level corresponding to 83.2 ag, which was more than1000 times the level detected in the remaining 15 normal tissues.

The expression of NY-SAR-35 mRNA was also examined in 26 sarcomaspecimens of various histologies, and was detected in fibrosarcoma andrhabdomyosarcoma specimens (2/26), as well as the SW1045 synovialsarcoma cell line (Table 7 and FIG. 1C). With regard to other tumortypes, transcripts encoding NY-SAR-35 were detected in 1/16 (6%)melanoma specimens, 5/29 (21%) lung cancer specimens, and 3/13 (23%)breast cancer specimens. NY-SAR-35 mRNA was not detected in small numberof colon cancer specimens (0/9) or in small numbers of renal cancerspecimens (0/8). Thus, on the basis of its immunogenicity in cancerpatients, and its restricted mRNA expression profile, NY-SAR-35 can beconsidered a novel CT antigen.

TABLE 7 Expression of NY-SAR-35 in sarcoma, sarcoma cell lines and othermalignant tissues Expression Histology Frequency Sarcomas Synovialsarcoma 0/8 Leimyosarcoma 0/4 Malignant Fibrous Histocytoma 0/4 EwingSarcoma 0/2 Osteosarcoma 0/2 Rhabdomyosarcoma 1/1 Fibrosarcoma 1/1Liposarcoma 0/1 Neurosarcoma 0/1 Chondrosarcoma 0/1 DFSP 0/1 SW1045synovial sarcoma cell positive line SW982 synovial sarcoma cell negativeline Fuji synovial sarcoma cell line negative Other MalignanciesMelanoma  1/16 Lung Cancer  5/29 Colon Cancer 0/9 Breast Cancer  3/13Renal Cancer 0/8 Esophageal Cancer  1/12 Ovarian Cancer  1/12 GastricCancer 5/6The NY-SAR-35 Gene, Transcript and Putative Protein and Orthologous Gene

An analysis of the human genome database, mapped the NY-SAR-35 cDNAsequence to Xq28, approximately 5.9 Mbp downstream (3′) of theCT10/MAGE-E1 gene and 6.8 Mbp upstream (5′) of the NY-ESO-1 gene. TheNY-SAR-35 gene is approximately 44 kb in length and spans 6 exons.Analyses of the human genome databases (NCBI GenBank,ncbi.nlm.nih.gov/genome, and Celera Genomics, Rockville, Md.,celera.com) revealed no genomic sequences of high similarity, suggestingthat it is a single copy gene with no additional family members. Theseresults were verified by probing Southern blots of human genomic DNAwith the NY-SAR-35 cDNA.

The present SEREX immunoscreening provided 4 overlapping NY-SAR-35 cDNAclones, ranging from 677-767 bp in length, all contained identical 3′sequences originating from the poly A region. The NY-SAR-35 cDNAsequence was identical to 3 ESTs (GenBank accession nos. AA909915,AA906131, and AW593050) which were all derived from the NFL_T_GBC_S1mixed tissue (fetal lung, testis, germinal center B cell) cDNA libraryand found in UniGene cluster Hs.128580 as well as 4 ESTs (GenBankaccession nos. BC034320, AK098602, BG771667 and BI465380) derived from atestis cell line and found in Unigene cluster Hs.375082. As shown inFIG. 1D, Northern blot analysis revealed a single NY-SAR-35 mRNAtranscript of 1.1 kB in normal testis, indicating the SEREX-definedclones and EST sequences represent partial transcripts. To obtain afull-length NY-SAR-35 transcript, 5′ RACE was performed, yielding 262 bpof additional 5′ DNA sequence. Thus, the total length of the NY-SAR-35transcript is 1029 bp (SEQ ID NO: 10, GenBank accession no. AY211917), asize that is in agreement with the 1.1 kb hybridization signal seen inNorthern blots of testis mRNA probed with NY-SAR-35 cDNA.

The NY-SAR-35 transcript encodes an open reading frame of 255 aminoacids (SEQ ID NO: 55, bp 68-895) with a predicted molecular mass of 29.2kDa. It is identical to a hypothetical protein, XM098959, predicted fromGenefinder analysis of human chromosome X sequences. The putativeNY-SAR-35 protein has a signal peptide, a transmembrane domain and acysteine-rich trefoil/P-domain, found in several secreted proteins ofthe gastrointestinal tract (Hoffmann W, Hauser F. The P-domain ortrefoil motif: a role in renewal and pathology of mucous epithelia?Trends Biochem Sci 1993 Jul.; 18(7):239-43). These data suggest thatNY-SAR-35 is a-secreted or membrane bound protein.

To identify a murine orthologue of NY-SAR-35, the putative humanNY-SAR-35 protein sequence was used to search a translated nonredundantnucleotide database by using the TBLASTN tool of the NCBI(ncbi.nlm.nih.gov/blast/Blast.cgi). A hypothetical mouse protein, termedXP_(—)150408, generated from a conceptual translation of the mouse Xchromosome, was found to have 57% identity (49/85 amino acids) withNY-SAR-35. Using nucleotide primers corresponding to sequences encodingXP_(—)150408, 5′ and 3′ RACE reactions were undertaken by using mousetestis cDNA. By combining 5′ and 3′ RACE products, a 1,202 bp cDNA wasidentified (GenBank accession no. AY214130, SEQ ID NO: 133). This cDNAencoded a putative full length mouse protein of 238 amino acids (SEQ IDNO: 134) which is 41% identical to human NY-SAR-35, with conservation ofthe trefoil and transmembrane domains. This murine NY-SAR-35(mNY-SAR-35) cDNA sequence was used to search mouse genome sequences(ncbi.nlm.nih.gov/genome/seq/MmBlast.html) yielding an identical genomesequence, NW 042622, from mouse chromosome X. Analysis of this sequenceshowed the mNY-SAR-35 gene is composed of approximately 42,600nucleotides and seven exons.

Example 2 Analysis of the NY-SAR-35 Protein and its Expression

Purification of Recombinant NY-SAR-35 Protein in E. Coli to ProduceMonoclonal Antibodies and to Perform ELISA Assays

There are four ATG codons in exon 1 of the NY-SAR-35 gene. It isexpected that the fourth ATG codon in the full length sequence ofNY-SAR-35 is the first ATG codon of the translated NY-SAR-35 sequence.It appears then that the predicted protein has two interesting domains.The protein revealed two distinctive hydrophobic domains followed by twohydrophilic turns. One hydrophobic domain is a signal peptide, which arepredicted in proteins with cleavage sites between amino acids 25 and 26with SignalP software tool available at the websitecbs.dtu.dk/services/SignalP. The other hydrophobic region is predictedto be a transmembrane domain with the TMHMM2.0 program available at thewebsite cbs.dtu.dk/services/TMHMM/TMHMM2.0b.guide.html. Therefore, theNY-SAR-35 gene encodes a signal peptide and a transmembrane domain (FIG.2).

Three kinds of NY-SAR-35 vectors were designed for the purification ofthe proteins in an E. coli expression system (pET System) (Novagen,Madison, Wis.). The first encoded the largest possible NY-SAR-35 proteinfrom the first ATG codon (SEQ ID NO: 150), the second encoded theNY-SAR-35 protein from the fourth ATG codon (MH7) (SEQ ID NO: 152), andthe third encoded the expected extracellular domain from the fourth ATGcodon (SEQ ID NO: 154). An illustration of these vectors is providedbelow. The expected sizes of the resulting proteins are 29 kD (263 aminoacids) (SEQ ID NO: 151), 22 kD (201 amino acids) (SEQ ID NO: 153) and14.6 kD (133 amino acids) (SEQ ID NO: 155), respectively.

I. Whole protein(NY-SAR-35 from the first ATG) Vector:pET23a(NdeI/XhoI):C-terminal His tag vector Primer; SAR35/NdeI: (SEQ ID NO: 156)CACACACACATATGTCTTCACATAGGAGGAAAGCGAAG SAR35/XhoI: (SEQ ID NO: 157)CACACACTCGAGCTCGTCACCATGTTCCTCACGTC (SEQ ID NO: 150)CATATGTCTTCACATAGGAGGAAAGCGAAGGGGAGGAATAGGAGAAGTCACCGTGCCATGCGTGTGGCTCACTTAGAGCTGGCAACTTATGAGTTGGCGGCAACTGAGTCGAATCCCGAGAGCAGCCATCCTGGATACGAGGCCGCCATGGCTGACAGGCCTCAGCCAGGATGGCGGGAATCTCTAAAGATGCGGGTCAGCAAACCCTTTGGGATGCTCATGCTGTCCATTTGGATCCTGCTGTTCGTGTGCTACTACCTGTCCTACTACCTGTGCTCCGGGTCCTCATATTTTGTGCTTGCAAATGGACATATCCTGCCCAACAGTGAAAATGCTCATGGCCAATCTCTGGAAGAAGATTCCGCATTGGAAGCTTTGCTGAATTTTTTCTTTCCAACAACTTGCAATCTGAGGGAAAATCAGGTGGCAAAGCCTTGTAATGAGCTGCAAGATCTTAGTGAGAGTGAATGTTTGAGACACAAATGCTGTTTTTCATCATCGGGGACCACGAGCTTCAAATGTTTTGCTCCATTTAGAGATGTGCCTAAACAGATGATGCAAATGTTTGGGCTTGGTGCGATCAGCCTTATCCTGGTATGTCTGCCCATTTATTGCCGCTCTCTTTTCTGGAGGAGCGAACCGGCCGATGATTTACAAAGGCAGGACAACAGAGTTGTAACGGGTTTGAAGAAACAAAGAAGGAAGCGAAAGAGGAAGTCTGAAATGTTACAGAAAGCAGCAAGAGGACGTGAGGAACATGGTGACGAGCTCGAG CACCACCACCACCACCACTGA (SEQ ID NO: 151)MSSHRRKAKGRNRRSHRAMRVAHLELATYELAATESNPESSHPGYEAAMADRPQPGWRESLKMRVSKPFGMLMLSIWILLFVCYYLSYYLCSGSSYFVLANGHILPNSENAHGQSLEEDSALEALLNFFFPTTCNLRENQVAKPCNELQDLSESECLRHKCCFSSSGTTSFKCFAPFRDVPKQMMQMFGLGAISLILVCLPIYCRSLFWRSEPADDLQRQDNRVVTGLKKQRRKRKRKSEMLQKAARGRE EHGDELEHHHHHH II.Partial protein(MH7 from the fourth ATG) Vector; pET23a(NdeI/XhoI)Primer; MH7/NdeI: (SEQ ID NO: 158) CACACACA CATATGCGGGTCAGCAAACCCTTTGGGASAR35/XhoI: (SEQ ID NO: 159) CACACA CTCGAGCTCGTCACCATGTTCCTCACGTC (SEQID NO: 152) CATATGCGGGTCAGCAAACCCTTTGGGATGCTCATGCTCTCCATTTGGATCCTGCTGTTCGTGTGCTACTACCTGTCCTACTACCTGTGCTCCGGGTCCTCATATTTTGTGCTTGCAAATGGACATATCCTGCCCAACAGTGAAAATGCTCATGGCCAATCTCTGGAAGAAGATTCCGCATTGGAAGCTTTGCTGAATTTTTTCTTTCCAACAACTTGCAATCTGAGGGAAAATCAGGTGGCAAAGCCTTGTAATGAGCTGCAAGATCTTAGTGAGAGTGAATGTTTGAGACACAAATGCTGTTTTTCATCATCGGGGACCACGAGCTTCAAATGTTTTGCTCCATTTAGAGATGTGCCTAAACAGATGATGCAAATGTTTGGGCTTGGTGCGATCAGCCTTATCCTGGTATGTCTGCCCATTTATTGCCGCTCTCTTTTCTGGAGGAGCGAACCGGCCGATGATTTACAAAGGCAGGACAACAGAGTTGTAACGGGTTTGAAGAAACAAAGAAGGAAGCGAAAGAGGAAGTCTGAAATGTTACAGAAAGCAGCAAGAGGACGTGAGGAACATGGTGACGAGCTCGAGCACCACCACCAC CACCACTGA (SEQ ID NO:153) MRVSKPFGMLMLSIWILLFVCYYLSYYLCSGSSYFVLANGHILPNSENAHGQSLEEDSALEALLNFFFPTTCNLRENQVAKPCNELQDLSESECLRHKCCFSSSGTTSFKCFAPFRDVPKQMMQMFGLGAISLILVCLPIYCRSLFWRSEPADDLQRQDNRVVTGLKKQRRKRKRKSEMLQKAARGREEHGDELEHHHH HH III. Expectedextracellular domain of NY-SAR-35 from the fourth ATGVector:pET23a(NdeI/XhoI) Primer; MH7/NdeI: (SEQ ID NO: 160) CACACACACATATGCGGGTCAGCAAACCCTTTGGGA MH7/XhoI: (SEQ ID NO: 161) CACACACTCGAGCATTTGCATCATCTGTTTAGGC (SEQ ID NO: 154)CATATGCGGGTCAGCAAACCCTTTGGGATGCTCATGCTCTCCATTTGGATCCTGCTGTTCGTGTGCTACTACCTGTCCTACTACCTGTGCTCCGGGTCCTCATATTTTGTGCTTGCAAATGGACATATCCTGCCCAACAGTGAAAATGCTCATGGCCAATCTCTGGAAGAAGATTCCGCATTGGAAGCTTTGCTGAATTTTTTCTTTCCAACAACTTGCAATCTGAGGGAAAATCAGGTGGCAAAGCCTTGTAATGAGCTGCAAGATCTTAGTGAGAGTGAATGTTTGAGACACAAATGCTGTTTTTCATCATCGGGGACCACGAGCTTCAAATGTTTTGCTCCATTTAGAGATGTGCCTAAACAGATGATGCAAATGCTCGAG CACCACCACCACCACC ACTGA (SEQ ID NO:155) MRVSKPFGMLMLSIWILLFVCYYLSYYLCSGSSYFVLANGHILPNSENAHGQSLEEDSALEALLNFFFPTTCNLRENQVAKPCNELQDLSESECLRHKCCFSSSGTTSFKCFAPFRDVPKQMMQMLEHHHHHH

Protein expression was induced in E. coli. Three colonies of each domaincloned plasmid were selected and cultured by IPTG induction for 4 hours.When total proteins were separated by SDS-electrophoresis and stained bySimply Blue SafeStain (Invitrogen) the highly expressed protein bandswere not detected. However, when total proteins, separated bySDS-polyacrylamide gel, were immunoblotted using an anti-His epitopeantibody, the His-tagged NY-SAR-35 proteins were detected. The resultsare shown in FIG. 3 with the expected sizes of the expressed proteins.

Functional Study of NY-SAR-35

Most cancer-testis antigens (11/13) have been found to be expressed innon-malignant human kidney embryonic 293 cell while NY-SAR-35 is not.Human 293 cell and monkey Cos-1 cells were used to stably express theNY-SAR-35 gene for functional and immunolocalization studies.

The expected NY-SAR-35 open reading frame (including the 5′ untranslatedregion) was cloned into pcDNA3.1/V5-HisA vector which had a C-terminalfusion tag (V5 epitope and 6×His epitope). The cloned NY-SAR-35nucleotide sequence and expected amino acid sequence are as follows:

A. Cloned NY-SAR-35 nucleotide sequence (SEQ ID NO: 162) GAATTCCTTCTGGGCCACGGACTGCCGGACCGTTGGGCTGTGAGGCAGCG EcoRITCTCAGCGAGGCGGCACCCGGAGCCATGTCTTCACATAGGAGGAAAGCGAAGGGGAGGAATAGGAGAAGTCACCGTGCCATGCGTGTGGCTCACTTAGAGCTGGCAACTTATGAGTTGGCGGCAACTGAGTCGAATCCCGAGAGCAGCCATCCTGGATACGAGGCCGCCATGGCTGACAGGCCTCAGCCAGGATGGCGGGAATCTCTAAAGATGCGGGTCAGCAAACCCTTTGGGATGCTCATGCTCTCCATTTGGATCCTGCTGTTCGTGTGCTACTACCTGTCCTACTACCTGTGCTCCGGGTCCTCATATTTTGTGCTTGCAAATGGACATATCCTGCCCAACAGTGAAAATGCTCATGGCCAATCTCTGGAAGAAGATTCCGCATTGGAAGCTTTGCTGAATTTTTTCTTTCCAACAACTTGCAATCTGAGGGAAAATCAGGTGGCAAAGCCTTGTAATGAGCTGCAAGATCTTAGTGAGAGTGAATGTTTGAGACACAAATGCTGTTTTTCATCATCGGGGACCACGAGCTTCAAATGTTTTGCTCCATTTAGAGATGTGCCTAAACAGATGATGCAAATGTTTGGGCTTGGTGCGATCAGCCTTATCCTGGTATGTCTGCCCATTTATTGCCGCTCTCTTTTCTGGAGGAGCGAACCGGCCGATGATTTACAAAGGCAGGACAACAGAGTTGTAACGGGTTTGAAGAAACAAAGAAGGAAGCGAAAGAGGAAGTCTGAAATGTTACAGAAAGCAGCAAGAGGACGTGAGGAACATGGTGACGAG CTCGAG TCTA                                        XhoI GAGGGCCCTTCGAA

               V5 epitope

CGTACCGGTCAT

TGA                   His tag B. Expected amino acid sequence andexpected size of expressed proteins (SEQ ID NO: 163)                        → 31 Kd            → 29 KdEFLLGHGLPDRWAVRQRLSEAAPGAMSSHRRKAKGRNRRSHRAMRVAHLE                       → 26 Kd       → 24 KdLATYELAATESNPESSHPGYEAAMADRPQPGWRESLKMRVSKPFGMLMLSIWILLFVCYYLSYYLCSGSSYFVLANGHILPNSENAHGQSLEEDSALEALLNFFFPTTCNLRENQVAKPCNELQDLSESECLRHKCCFSSSGTTSFKCFAPFRDVPKQMMQMFGLGAISLILVCLPIYCRSLFWRSEPADDLQRQDNRVVTGLKKQRRKRKRKSEMLQKAARGREEHGDELESRGPFEGKPIPNPLLGLD                                      V5 epitope STRTGHHHHHH      Histag

Human 293 cells and monkey Cos-1 cell that stably express the NY-SAR-35gene were tested by RT-PCR and Western blotting. The cells transfectedwith 0.5 μg pcDNA3.1/V5/HisA/NY-SAR-35 plasmid were selected with 1mg/ml neomycin for 14 days. Clones were picked and expanded for anadditional 1 month and analyzed for NY-SAR-35 mRNA and proteinexpression. Three sets of NY-SAR-35 5′/3′ primers were used and areprovided below:

lane 1 (ORF including 5′ untranslated region) (SEQ ID NO: 164)GGGAATTCATGTCTTCACATAGGAGGAAAGCG/CACACACTCGAGCTCGT CACCATGTTCCTCACGTClane 2 (ORF from the first ATG) (SEQ ID NO: 165)CACACACACATATGTCTTCACATAGGAGGAAAGCGAAG/CACACACTCGAGCTCGTCACCATGTTCCTCACGTC lane 3 (ORF from the fourth ATG) (SEQ ID NO:166) CACACACACATATGCGGGTCAGCAAACCCTTTGGGA/CACACACACATATGTCTTCACATAGGAGGAAAGCGAAG lane 4 (p53 5′/3′) (SEQ ID NO: 167)TACTCCCCTGCCCTCAACAAG/CTCAGGCGGCTCATAGGG

Whole cell extracts were made from the same cloned cells. Total proteinswere separated by SDS-polyacrylamide gel electrophoresis andimmunoblotted to detect NY-SAR-35 proteins by anti-V5 epitope monoclonalantibody (Invitrogen). The size of the stably expressed NY-SAR-35proteins in 293 and Cos-1 cells was found to be 24 kD. This is,therefore, consistent with translation of NY-SAR-35 beginning at thefourth ATG.

Example 3 Results from the Second Round of Immunoscreenings by SEREXAnalysis

Identification of Human Sarcoma Antigens by SEREX Analysis

Serum from the two NY-ESO-1 seropositive patients (FS and MFH) wereagain used to immunoscreen cDNA libraries prepared from the SW982 andSW1045 synovial sarcoma cell lines, both of which were shown to expresseight or more known CT antigen transcripts (Table 2). Sera from the FSpatient was also used to immunoscreen a cDNA library derived from normaltestis. In total, the results from Examples 1 and 3 represent fiveindependent SEREX immunoscreenings performed, which lead to theidentification of 113 distinct antigens, designated NY-SAR-1 throughNY-SAR-113.

The 113 SEREX-defined antigens represent 91 known proteins and 22uncharacterized gene products (novel, ESTs, KIAA series, FLJ series,ORFs, DKFZ series). In addition to the uncharacterized gene productsdescribed above in Example 1 (NY-SAR-3, -10, -16, -22, -23, -24, -27,-29, -35, -41, -48 and -71) additional immunoscreening identifiedanother 11 uncharacterized gene products (NY-SAR-77, -79, -80, -84, -88,-91, -95, -97, -104, -105 and -113). All of the sequences for theseuncharacterized gene products have been deposited in the GenBankdatabase and given the sequential accession numbers AY211909-AY211931.In terms of the serum sources, 27 of the 113 antigens were identified byusing sera from a MFH patient and 86 were identified with FS sera. Ofthe 113 antigens identified, 95 were unique to a particular cDNA libraryscreening and 18 antigens were identified in more than one library. Thisunderlines the beneficial nature of incorporating multiple cDNAlibraries into large-scale SEREX analyses of the cancer immunome.

Seroepidemiology of SEREX-defined Sarcoma Antigens

The cDNA sequences encoding the 113 sarcoma antigens were compared withsequences deposited in the cancer immunome or SEREX databaselicr.org/CancerImmunomeDB, formerly licr.org/SEREX.html). Thesecomparisons are in addition to the comparisons presented above inExample 1. In a preliminary analysis, it was found that 39 of the 113sarcoma antigens defined in this study (34%) were also identifiedthrough SEREX analysis of other tumor types (Table 8). Table 9 belowprovides a complete list of all 113 antigens along with their respectiveUnigene cluster information, if any. These results represent theinformation available after all rounds of immunoscreening. Contrary tothe results shown, NY-SAR-39, -57, -61, -63 and -64 after the firstround of immunoscreenings had not been found in the SEREX database.

TABLE 8 Immunomic analysis of sarcoma/testis antigens: Reactivity withsera from sarcoma patients, patients with other forms of cancer, andnormal individuals Gene identity Cancer patient Normal NY-SAR-antigen(ugene cluster) seroreactivity* seroreactivity 1 TMF1 (Hs.267632) GC,BC, CC, SRC 2/33 2 STAU (Hs.6113) PC, BC, SRC 3/30 3 KIAA1536(Hs.156667) BC, SRC 2/33 6 RHAMM (Hs.72550) OC, SRC 1/33 7 PINCH(Hs.112378) CC, GC, RC, BC HN, 16/21  ESO, AML, SRC 11 U2AF1RS2(Hs.171909) RC, HD, BC, GC, SRC 6/33 13 ACTN1 (Hs.119000) BC, SRC 5/3015 RBM6 (Hs.173993) LC, SRC 0/33 16 FLJ12785 (Hs.192742) TALL, SRC 0/3317 LAGE-1a (Hs.87225) BC, SRC 0/33 18 SSSCA1(Hs.25723) CC, SRC 0/33 19HEF1 (Hs.80261) RC, SRC 3/33 28 PPIL4 (Hs.11065) BC, SRC 0/33 29FLJ13441 (Hs.232146) PN, SRC 6/33 31 AUANTIG (Hs.75528) BC, GC, OC, SRC2/33 39 PSMD4 (Hs.148495) MEL, SRC 0/33 44 LGALS1 (Hs.227751) RC, SRC0/33 45 STIP1 (Hs.75612) RC, SRC 4/33 47 MIF (Hs.73798) MEL, SRC 0/33 57GCN5L2 (Hs.101067) PC, SRC 0/33 61 ZNF282 (Hs.58167) RC, SRC 1/33 63USP19 (Hs.301373) OC, SRC 0/33 64 USP16 (Hs.99819) PN, SRC 2/33 66 ROCK1(Hs.17820) RC, BC, CC, SRC 1/33 74 RANBP2 (Hs.199179) BC, GL, BC, SRC2/33 77 KIAA0992 (Hs.194431) PC, SRC 4/15 80 FLJ12577 (Hs.87159) GC, SRC0/33 81 SDS3 (Hs.20104) GC, SRC 4/16 82 NYCO45 (Hs.160881) CC, SRC 0/3389 SSX2 (Hs.289105) BC, MEL, SRC 0/33 90 UACA (Hs.49753) BC, ESO, SRC4/25 93 NYBR15 (Hs.178175) BC, SRC 1/12 98 OIP2 (Hs.274170) BC, SRC 0/3399 SSX3 (Hs.178749) BC, MEL, SRC 2/30 101 RANBP2L1 (Hs.179825) GL, BC,SRC 3/33 102 RBPJK (Hs.356806) GC, RC, BC, MEL, 1/16 SRC 103 Hsp40(Hs.94) HN, NCC, SRC 0/33 108 EIF4G (Hs.25732) GC, SRC 5/27 112 PMSCL1(Hs.91728) CC, SRC 0/33 AML, acute myelogenous leukemia; BC, breastcancer; CC, colon cancer; GC, gastric cancer; GL, glioma; HCC,hepatocellular carcinoma; HN, head and nech cancer; LC, lung cancer;MEL, melanoma; OC, ovarian cancer; PC, prostate cancer; PN, pancreaticcancer; RC, renal cancer; SRC, sarcoma; TALL, T cell acute lymphocyticleukemia. *Determined by sequence comparisons with the SEREX database(licr.org/CancerImmunomeDB/).

TABLE 9 Sarcoma/testes antigens defined by serological analysis of cDNAexpression libraries NY- SAR- Gene identity Sera Library antigen(Unigene Cluster) source source 1 TMF1 (Hs.267632) MFH, FS A, T 2 STAU(Hs.6113) MFH A 3 KIAA1536 MFH A (Hs.156667) 4 FH (Hs.75653) MFH, FS A,B 5 TBC1D1 (Hs.278586) MFH A 6 RHAMM MFH A, B (Hs.72550) 7 PINCH(Hs.112378) MFH A, B 8 BIRC2 (Hs.289107) MFH A, B 9 ATP5B (Hs.25) MFH A,B 10 KIAA0603 MFH A (Hs.173802) 11 U2AF1RS2 MFH A, B (Hs.171909) 12NESG1 (Hs.158450) MFH B 13 ACTN1 (Hs.119000) MFH A 14 SC65 (Hs.207251)MFH A 15 RBM6 (Hs.173993) MFH A 16 FLJ12785 MFH A (Hs.192742) 17 LAGE-1aMFH, FS B (Hs.87225) 18 SSSCA1 (Hs.25723) MFH A, B 19 HEF1 (Hs.80261)MFH A, B 20 TCEB3 (Hs.155202) MFH B 21 GTF3C3 (Hs.90847) MFH A 22 NELIN(Hs.216381) MFH A 23 C20orf81 MFH A (Hs.29341) 24 None (not clustered)MFH A 25 PDE4DIP MFH B (Hs.265848) 26 PIASX-BETA MFH B (Hs.111323) 27FLJ10330(Hs.342307) MFH B 28 PPIL4 (Hs.11065) FS B 29 FLJ13441 FS A(Hs.232146) 30 SNK (Hs.3838) FS A 31 HUMAUANTIG FS A, B, T (Hs.75528) 32PDAP1 (Hs.278426) FS A 33 SURF6 (Hs.274430) FS B 34 SEC23B FS B(Hs.173497) 35 EST (Hs.128580) FS B, T 36 SSX1(Hs.194759) FS B, T 37 MP1(Hs.260116) FS A, T 38 HMG20B FS A (Hs.32317) 39 PSMD4 (Hs.148495) FS A40 INPP1 (Hs.32309) FS A 41 EST (Hs.166670) FS B 42 BTG3 (Hs.77311) FSB, T 43 SSX4 (Hs.278632) FS B 44 LGALS1 FS B (Hs.227751) 45 STIP1(Hs.75612) FS A 46 ARNTL2 FS B (Hs.222024) 47 MIF (Hs.73798) FS A 48MGC20533 FS A (Hs.69280) 49 EMK1 (Hs.157199) FS A 50 PYCR1 (Hs.79217) FSA 51 EDF1 (Hs.174050) FS A 52 Actin (Hs.288061) FS A 53 FXYD5(Hs.333418) FS A 54 LMOD1 (Hs.79386) FS A 55 RBM10 (Hs.154583) FS A 56MLF1(Hs.85195) FS A, T 57 GCN5L2 FS A (Hs.101067) 58 LIP8 (Hs.348012) FSA 59 UPF3B FS A (Hs.103832) 60 EGLN1 (Hs.6523) FS A 61 ZNF282 FS A(Hs.58167) 62 AD034(Hs.281397) FS A 63 USP19(Hs.301373) FS A 64 USP16(Hs.99819) FS B, T 65 FDFT1 (Hs.48876) FS B 66 ROCK1 FS B, T (Hs.17820)67 LUC7L FS B (Hs.16803) 68 P38IP (Hs.333500) FS B 69 ARL1 FS B(Hs.242894) 70 RPL10A FS B (Hs.334895) 71 EST (Hs.314941) FS B 72 HSPE1(Hs.1197) FS B, T 73 PRM2 (Hs.2324) FS T 74 RANBP2 FS T (Hs.199179) 75GKAP42 FS T (Hs.36752) 76 TIAL1 FS T (Hs.182741) 77 KIAA0992 FS T(Hs.194431) 78 TSP-NY FS T (Hs.97643) 79 Novel (not FS T clustered) 80FLJ12577 FS T (Hs.87159) 81 SDS3 (Hs.20104) FS T 82 NYCO45 FS T(Hs.160881) 83 SOX6 FS T (Hs.326876) 84 DKFZp434 FS T (Hs.131834) 85RAD50 FS T (Hs.41587) 86 EPIM (Hs.99865) FS T 87 SOX5 (Hs.87224) FS T 88DKFZp564 FS T (Hs.93589) 89 SSX2 (Hs.289105) FS T 90 UACA (Hs.49753) FST 91 FLJ11730 FS T (Hs.17118) 92 ESTs (Hs.368781) FS T 93 NYBR15 FS T(Hs.178175) 94 CG005 (Hs.23518) FS T 95 FLJ10637 FS T (Hs.22595) 96 MCSPFS T (Hs.111850) 97 EST (Hs.128836) FS T 98 OIP2 (Hs.274170) FS T 99SSX3 (Hs.178749) FS T 100 PGAM2 FS T (Hs.46039) 101 RANBP2L1 FS T(Hs.179825) 102 RBPJK FS T (Hs.356806) 103 Hsp40 (Hs.94) FS T 104DKFZp434 FS T (Hs.131834) 105 C11orf14 FS T (Hs.32017) 106 CEP11(Hs.97437) FS T 107 UBE1 (Hs.2055) FS T 108 EIF4G (Hs.25732) FS T 109SYNJ1 FS T (Hs.127416) 110 NYD-SP14 FS T (Hs.98105) 111 NDP52 FS T(Hs.154230) 112 PMSCL1 FS T (Hs.91728) 113 KIAA0442 FS T (Hs.32168)

To determine whether immune recognition of these 39 antigens wascancer-related, serum samples from normal individuals (n=33) were testedfor reactivity to these antigens. 23 of the 39 antigens (59%) had aserological profile that was not restricted to cancer patients, whereasthe remaining 16 antigens had a cancer-related serological profile,reacting only with sera from cancer patients (sarcoma patients and serumsource of SEREX database entry), and not with sera from normalindividuals. 14 of these 16 antigens reacted only with sera from asingle sarcoma patient when tested for reactivity with additionalallogeneic sarcoma sera (n=39). The remaining 2 antigens,NY-SAR-17/LAGE-1 and NY-SAR-80/FLJ12577, reacted with 2 of 39 and 3 of39 sarcoma sera, respectively, and not with sera from normal individuals(n=33).

NY-SAR-80/FLJ12577 is an uncharacterized member of the Mo25 proteinfamily, an evolutionary conserved family of proteins with no knownfunction. Analysis of the tissue distribution and frequency of ESTsequences homologous to NY-SAR-80/FLJ12577 indicate widespread mRNAexpression, with a preponderance of malignant tissue-derived homologousESTs suggesting possible overexpression in cancer.

Overall, the relative infrequency of overlapping humoral immuneresponses among the population of sarcoma patients analyzed is contraryto previous findings for colon (Scanlan M J. et al. 2002. Cancer-RelatedSerological Recognition of Human Colon Cancer: Identification ofPotential Diagnostic and Immunotherapeutic Targets. Cancer Res., 2002;Jul. 15; 62(14), 4041-7.), breast (Scanlan M J, et al. Humoral immunityto human breast cancer: antigen definition and quantitative analysis ofmRNA expression. Cancer Immunity 1:4 [epub]) and renal cancers (Scanlan,M. J., et al., and Old, L. J. Antigens recognized by autologous antibodyin patients with renal-cell carcinoma. Int. J. Cancer 1999; 83: 456-64)in which a subset of antigens were mutually seroreactive in a cancerrelated manner. These results suggest that the immune response tosarcoma is either highly variable or that distinct sarcoma histiotypeshave distinct immunomes.

Expression Patterns of mRNA Encoding Serologically DefinedSarcoma/Testis Antigens in Normal and Malignant Tissues

In addition to the three well-known CT antigens described in Example 1,NY-SAR-89/SSX-2 and NY-SAR-99/SSX-3 were found to have restricted ESTprofiles, being expressed exclusively in normal testis and a range ofdifferent tumor types (Lethe B, et al. 1998. LAGE-1, a new gene withtumor specificity. Int. J. Cancer 76:903-8; Türeci Ö, et al. 1998.Expression of SSX genes in human tumors. Int J. Cancer 77:19-23; GureAO, et al. 1997. SSX: a multigene family with several memberstranscribed in normal testis and human cancer. Int J Cancer 72:965-971).Six other putative tissue-restricted antigens were identified, includingfour other known gene products, NY-SAR-73/Protamine 2 (PRM2, Domenjoud,L., Fronia, C., Uhde, F. & Engel, W. (1998) Nucleic Acids Res. 16,7773), NY-SAR-78/TSP-NY (UniGene cluster Hs.97643),NY-SAR-96/mitochondrial capsule selenoprotein (MCSP, Aho, H., et al.(1996) Geonomics 32, 184-190) and NY-SAR-110/NYD-SP14 (Hs.98105) and twoadditional uncharacterized gene products, NY-SAR-92 (Hs.368781) andNY-SAR-97 (not clustered).

Two of the six putative tissue restricted antigens, NY-SAR-73/PRM2 andNY-SAR-110/NYD-SP14, were ubiquitously expressed in a panel of 20 normaltissues as determined by RT-PCR (Table 10). The remaining four genes, inaddition to NY-SAR-12/nasopharyngeal specific protein 1 (NESG1, Li Z,Yao K, Cao Y. Molecular cloning of a novel tissue-specific gene fromhuman nasopharyngeal epithelium. Gene 1999 Sep. 3; 237(1):235-40),NY-SAR-35 and NY-SAR-41, were found to be expressed with frequenciesranging from 1 to 9 of 20 normal tissues. NY-SAR-35 and NY-SAR-78 wereboth testis-specific. The mRNA expression profiles of NY-SAR-35 andNY-SAR-78 were then analyzed in various malignant tissues by RT-PCR.Transcripts encoding NY-SAR-78/TSP-NY were not detected in cancer. Thetumor specimens examined included, lung cancer (0 of 9), colon cancer (0of 9), breast cancer (0 of 18), renal cancer (0 of 11), esophagealcancer (0 of 12), ovarian cancer (0 of 14), melanoma (0 of 18) andsarcoma (0 of 8). Thus, although NY-SAR-78/TSP-NY is a “virtual CTantigen” with 100% identity with ESTs derived from prostate cancer andleukemia, its expression in cancer could not be verified in our RT-PCRseries.

TABLE 10 Analysis of mRNA expression by RT-PCR of 9 of the 113sarcoma/testis antigens NY-SAR antigen* Tissue 12 35 41 73 78 92 96 97110 Brain − − − + − − − − + Kidney − − − + − − − − + Liver − − − + − − −− + Pancreas − − − + − − − − + Placenta + − − + − − − − +Testis + + + + + + + + + Fetal brain − − + + − − + + + Small − − − + − −− − + intestine Heart − − − + − − − − + Prostate − − − + − − + + +Adrenal − − − + − − + + + Spleen + − − + − + + + + Colon + − + + − − −− + Stomach − − − + − − − − + Lung + − + + − − − + + Bladder − − + + − −− + + Ovary + − + + − − − + + Breast − − − + − − − + + Cervix − − − + −− − − + Skeletal − − − + − − − − + muscle Total no. 6/ 1/20 6/20 20/201/20 2/20 5/20 9/20 20/20 of positive 20 tissues *Unigene clusters:NY-SAR-12, Hs.158450; NY-SAR-35, Hs.128580; NY-SAR-41, Hs.166670;NY-SAR-73, Hs.2324; NY-SAR-78, Hs.97643; NY-SAR-92, Hs.368781;NY-SAR-96, Hs.111850; NY-SAR-97, Hs.128836; NY-SAR-110, Hs.98105.

The antigens presented herein are of interest for theirimmunotherapeutic and diagnostic potential. For example, the six knowntestis-restricted gene antigens (NY-SAR-17/LAGE-1, NY-SAR-36/SSX1,NY-SAR-43/SSX4, NY-SAR-78/TSP-NY, NY-SAR-89/SSX2 and NY-SAR-99/SSX3),four novel gene products that are also differentially expressed antigens(NY-SAR-35, 41, -92 and -91) and two tissue-restricted antigens(NY-SAR-12/NESG1 and NY-SAR-96/MCSP) not previously studied in relationto cancer have are potential vaccine targets and/or targets fortherapeutic antibodies as well as for diagnosis of cancer, particularlyby screening patient samples for antibodies that recognize the proteins.

NY-SAR-35 mRNA was detected in a variety of tumor specimens, such asmelanoma (1 of 16 specimens), sarcoma (2 of 26 specimens), lung cancer(5 of 29 specimens), breast cancer (3 of 13 specimens), bladder cancer(5 of 12 specimens), esophageal cancer (1 of 12 specimens) and ovariancancer (1 of 12 specimens). As also shown before in Example 1, NY-SAR-35was not detected in colon cancer (n=9) or renal cancer (n=8). TheCT-restricted expression profile of NY-SAR-35 was confirmed by real-timequantitative RT-PCR at 40 amplification cycles (FIG. 4). In two of thenine non-small lung cancer specimens tested, NY-SAR-35 was expressed atlevels that were 0.13 and 0.15 times the level detected in normaltestis. In conformity with the proposed nomenclature for CT antigens(Chen Y T, et al. 1998. Identification of multiple cancer/testisantigens by allogeneic antibody screening of a melanoma cell linelibrary. Proc. Natl. Acad. Sci. USA. 95:6919-23), NY-SAR-35 isdesignated CT-20.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1. An isolated polypeptide consisting of a sequence as set forth as SEQID NO: 55 or a fragment thereof that is at least 8 amino acids inlength.
 2. The isolated polypeptide of claim 1, wherein the fragment isat least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 75 or 100 amino acids in length.
 3. A composition comprising theisolated polypeptide of claim 1 or 2 and a pharmaceutically acceptablecarrier.
 4. The composition of claim 3, wherein the composition furthercomprises an adjuvant.
 5. The composition of claim 3, wherein thecomposition further comprises a cytokine.
 6. The composition of claim 3,wherein the composition further comprises a costimulatory molecule. 7.The composition of claim 3, wherein the composition further comprises aMUC molecule, and wherein the isolated polypeptide is complexed to theMHC molecule.
 8. The composition of claim 7, wherein the MHC molecule isa HLA class I molecule.
 9. The composition of claim 7, wherein the MHCmolecule is a HLA class II molecule.
 10. A kit for detecting antibodiesreactive to a sarcoma-associated antigen in a biological sample,comprising: one or more sarcoma-associated antigens consisting of asequence as set forth as SEQ ID NO: 55 or a fragment thereof that is atleast 8 amino acids in length, and instructions for the use of thesarcoma-associated antigens in the detection of antibodies in thebiological sample.
 11. The kit of claim 10, wherein the fragment is atleast 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 75 or 100 amino acids in length.
 12. The kit of claim 10 or 11,wherein the sarcoma-associated antigen is bound to a substrate.
 13. Anisolated polypeptide comprising a sequence as set forth as SEQ ID NO: 55or a fragment thereof that is at least 45 amino acids in length.
 14. Theisolated polypeptide of claim 13, wherein the fragment is at least 50,75 or 100 amino acids in length.
 15. A composition comprising theisolated polypeptide of claim 13 or 14 and a pharmaceutically acceptablecarrier.
 16. The composition of claim 15, further comprising anadjuvant, a cytokine or a costimulatory molecule.
 17. The composition ofclaim 15, wherein the composition further comprises a MHC molecule, andwherein the isolated polypeptide is complexed to the MHC molecule. 18.The composition of claim 17, wherein the MHC molecule is a HLA class IImolecule.
 19. A kit for detecting antibodies reactive to asarcoma-associated antigen in a biological sample, comprising: one ormore sarcoma-associated antigens comprising a sequence as set forth asSEQ ID NO: 55 or a fragment thereof that is at least 45 amino acids inlength, and instructions for the use of the sarcoma-associated antigensin the detection of antibodies in the biological sample.
 20. The kit ofclaim 19, wherein the fragment is at least 50, 75 or 100 amino acids inlength.
 21. The kit of claim 19 or 20, wherein the sarcoma-associatedantigen is bound to substrate.
 22. A composition comprising an isolatedpolypeptide comprising (a) a sequence as set forth as SEQ ID NO: 55 or afragment thereof that is at least 8 amino acids in length, (b) a MHCmolecule and (c) a pharmaceutically acceptable carrier, wherein theisolated polypeptide is complexed to the MUC molecule.
 23. Thecomposition of claim 22, wherein the fragment is at least 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75 or 100 aminoacids in length.
 24. The composition of claim 22 or 23, wherein the MHCmolecule is a HLA class I molecule.
 25. The composition of claim 22 or23, wherein the MHC molecule is a HLA class II molecule.
 26. Acomposition comprising an isolated polypeptide comprising (a) a sequenceas set forth as SEQ ID NO: 55 or a fragment thereof that is at least 8amino acids in length, (b) an adjuvant, cytokine, or a costimulatorymolecule, and (c) a pharmaceutically acceptable carrier.
 27. Thecomposition of claim 26, wherein the fragment is at least 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75 or 100 aminoacids in length.